Fatty acids, C18 unsat, reaction products with diethylenetriamine

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-02-22 to 2010-08-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP, All validity criteria fulfilled, complete identification of test substance, including chemical analyses

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Two concentration levels 30.0 and 270 µg/L and the control were analysed at least once within 7 days in the fresh (0 h) and old media (48 h).
- Sampling method: For the longest exposure interval of 72 h samples were taken from the fresh (0 h) and old media (72 h) once within the test period, respectively. For the analyses of the old media additional replicates without algae and test organisms were prepared and stored under test conditions.
The sorption of the test item to the glassware was quantified once during the test period after the first sampling interval. Therefore, one test vessel of the test concentration 30.0 µg/L, containing daphnia and algae, was emptied after 72 h of exposure and rinsed twice with water. Thereafter an extraction of the test vessel was carried out.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/L was freshly prepared with dilution water and treated with ultrasound for 5 min at room temperature.
- Eluate: NAtural river water
- Differential loading: 10.0 - 30.0 - 90.0 - 270 - 810 µg/L
- Controls: 10 replicates of diulution water without test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Frequency: daily


ACCLIMATION
- Acclimation period: 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: During acclimation the daphnids were not fed
- Feeding frequency: None
- Health during acclimation (any mortality observed): Healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The parent animals were removed from the culture medium and the juveniles collected over a sieve and flushed into fresh medium

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 18 Day 21
Feb. 22nd Feb. 24th March 1st March 3rd March 12th March 15th
810 114 119 n.a. n.a. n.a. n.a.
270 -- -- 127 120 116 118
Control 100 100 76 116 112 112

Test temperature:

Temperature [°C]
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 18 Day 21
Feb. 22nd Feb. 24th March 1st March 3rd March 12th March 15th
810 21.9 21.1 n.a. n.a. n.a. n.a.
270 -- -- 21.2 21.7 19.4 21.4
Control 19.1 21.5
19.0 21.7 19.0 21.9

pH:

pH-Values
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 18 Day 21
Feb. 22nd Feb. 24th March 1st March 3rd March 12th March 15th
810 7.62 7.55 n.a. n.a. n.a. n.a.
270 -- -- 7.84 7.60 7.97 7.55
Control 8.07 7.79 8.07 7.70 8.11 7.59

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 18 Day 21
Feb. 22nd Feb. 24th March 1st March 3rd March 12th March 15th
810 8.57 5.71 n.a. n.a. n.a. n.a.
270 -- -- 8.85 7.60 9.39 7.51
Control 9.32 6.95 9.41 7.98 9.60 7.90

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to information in materials and methods incl. tables

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency): 3 x per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural river water of the river Leine was used. This river is located near D-31171 Nordstemmen, Germany.

Location D-31171 Nordstemmen
Sampling Date 2009-12-15

Weather conditions
on Day of Sampling Cloudy, ca -1 °C
Colour Yellowish, clear
pH 7.97
Conductivity [µS/cm] 386
Dissolved Oxygen [mg O2] 8.62
DOC [mg C/L] 3.9
TOC [mg C/L] 3.9
Ammonium-N [mg N/L] 0.042
Nitrate-N [mg N/L] 2.62
Total Nitrogen [mg N/L] 3.53
o-Phosphate-p [mg P/L] 0.062
Total Phosphate [mg P/L] 0.053
Suspended Matter [mg/L] 16.2
Total Hardness [mg CO3/L] 154
Storage conditions < 10 °C


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark per day
- Light intensity: Max. 20 µE*m-2*s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Adult mortality: daily
- Number of juveniles: daily
- Stillborn juveniles and aborted eggs: daily
- Appearance of first brood
- Intrinsic rate of natural increase: test end
- Growth (total length and dry weight): test end


RANGE-FINDING STUDY
- Test concentrations: 10 - 1 - 0.1 mg/L
Immobilization Rates [%] of the Preliminary Acute Immobilization Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item
Concentration
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicate Replicate
1 2 MV 1 2 MV
10 100 100 100 100 100 100
1 50 20 35 100 100 100
0.1 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

270 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

270 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Parental

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

311 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

parental

Remarks on result:

other: (270-810)

Details on results:

- Mortality of parent animals:
The test item induced significant adult mortality at the concentration level 810 µg/L (100 %) after 21 days. At the other concentration levels and in the control no biologically significant mortality (≤ 20 %) of parent animals was observed. The EC50 for adult mortality after 21 days was calculated to be 311 µg/L (confidence limits: 270- 810 µg/L).
- No. of offspring produced per day per female:
Four to five broods were released by all surviving animals of the control and concentration levels 10.0 to 270 µg/L.
The average number of juveniles per parent in the control group was 121 after 21 days. The reproductive output was statistically significant increased at the tested concentration level 270 µg/L when compared to the control (One Way Analysis of Variance, DUNNETT´S method p = 0.05). The reproductive outputs at the concentration levels 10.0 to 90.0 µg/L was comparable to the control group
The coefficient of variation of the mean number of living offspring produced per parent alive at the control group was calculated to be 11 %. At the concentration levels 10.0 to 270 µg/L the calculated coefficient of variation was in a range comparable to the control and is considered to be not biologically significant (coefficients of variation < 25 %).
- Body length and weight of parent animals:
The mean dry body weights of the surviving parental daphnids, determined at the end of the study, at the concentration levels 10.0 to 270 µg/L were in the range of 0.92 to 0.99 mg and comparable to the mean value of 0.91 mm per daphnid at the control group. The mean values of the total body length of the surviving parental daphnids at the concentration levels 10.0 to 270 µg/L were in the range of 5.58 to 5.63 mm per daphnid and comparable to the mean value of 5.55 mm per daphnid at the control group.
- Number of males and females (parental):
No males were observed in either the control or the test groups during the test.
- Time to first brood release or time to hatch:
The first day of appearance of juveniles at the tested concentration levels 10.0 to 270 µg/L and the control groups producing juveniles was between day 7 and day 8

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.44 (CI 1.35 - 1.54)

Reported statistics and error estimates:

Significant deviations were evaluated in comparison to the control using statistical standard procedures as Normality Test, Equal Variance Test and Analysis of Variance.
Statistical evaluation of the reproduction rates was carried out using One Way Analysis of Variance, DUNETT´S method (p = 0.05) and the statistical evaluation of the intrinsic rates of natural increase was carried out using One Way Analysis of Variance (p = 0.05). The coefficients of variation around the mean number of living offspring produced per parent in the control and the test groups were evaluated.
The estimation of the EC50-value for adult mortality and the reference item was carried out by using sigmoidal dose-response regression. The confidence intervals for the EC50-value of the reference item was calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism. For the EC50-value of adult mortality the highest concentration causing no effect and the lowest concentration producing 100 % were used as confidence limits. The estimation of an EC10- or EC50-value for the reduction or increase of the reproductive output could not be performed due to the absence of effects on reproduction.

Number of Juveniles alive in the Control and Test Groups after 21 Days

Nominal conc. [µg/L]												No.	Mean No. of Juveniles alive			Comparison
	Number of Juveniles alive in Replicate No.										Total	of			CV	versus
											No.	Parents	per Parent			Control
												prod. Juv.	producing Juveniles			Red.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
810	--	--	--	--	--	--	--	--	--	--	--	0	n.a.		n.a.	n.a.	n.a.
270	135	135	133	141	176	148	132	--	126	143	1269	9	141   ±	15	10	-16.5	yes
90.0	131	135	111	128	124	102	91	121	109	133	1185	10	119   ±	15	12	1.65	no
30.0	108	114	142	118	141	116	145	111	126	126	1247	10	125   ±	14	11	-3.31	no
10.0	134	125	92	133	111	118	131	126	134	125	1229	10	123   ±	13	11	-1.65	no
Control	95	150	118	126	118	118	117	128	125	112	1207	10	121   ±	14	11	—	—

First Appearance of Living Juveniles at the Individual Groups

Nominal concentration	Day of First Appearance of Living Juveniles										First
	in Replicate No.										Appearance
[µg/L]	1	2	3	4	5	6	7	8	9	10	Mean Day
810	--	--	--	--	--	--	--	--	--	--	--
270	8	7	8	8	7	8	7	--	7	8	7.44
90.0	7	8	7	8	7	8	10	7	8	8	7.80
30.0	8	8	8	8	8	8	8	8	8	8	8.00
0.10	8	8	10	8	7	8	8	9	8	8	8.20
Control	8	8	8	8	8	8	8	8	8	8	8.00

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure

                 (n = 10)

Nominal concentration	Adult Mortality [%]
[µg/L]	7 days	14 days	21 days
810	100*	100*	100*
270	0	0	10
90.0	0	0	0
30.0	0	0	0
10.0	0	0	0
Control	0	0	0

Total Body Length and Dry Weight of the Parent Animals

Nominal concentration [µg/L]	Total Length of the Parent Animals [mm]										MV	N	Dry Weight [mg]
	Replicate No.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
810	--	--	--	--	--	--	--	--	--	--	--	--	--	--
270	5.75	5.75	5.75	6.00	5.50	5.75	5.25	--	5.00	5.50	5.58	9	8.4	0.93
90.0	5.50	5.75	5.25	5.75	6.00	5.25	5.25	5.75	5.75	5.50	5.58	10	9.9	0.99
30.0	5.75	5.50	5.75	5.50	6.00	5.50	5.25	5.75	5.50	5.75	5.63	10	9.2	0.92
10.0	5.50	5.25	5.75	5.75	5.75	5.50	5.75	5.50	5.75	5.75	5.63	10	9.5	0.95
Control	5.75	5.50	5.75	5.50	5.50	5.75	5.50	5.25	5.50	5.50	5.55	10	9.1	0.91

Validity criteria fulfilled:

yes

Conclusions:

The adult mortality at the concentration level 810 µg/L was the most sensitive effect in this study. In this study no reduction of reproductive output, but hormesis was observed. The reproductive output was statistically significantly increased at 270 µg/L. The NOEC was assessed as adverse effect level directly from the observation data taking the observed hormesis not into account.
The estimation of an EC10- or EC50-value for the reduction or increase of the reproductive output could not be performed due to the absence of statistically significant effects on the reproduction. Effect values are as following:

Adverse effect value: NOECReproduction: 270 µg/L

Effect values: EC10, Reproduction : Not applicable
EC50, Reproduction : Not applicable

EC10, Adult Mortality : 270 µg/L
EC50, Adult Mortality : 311 µg/L (confidence limits: 270 - 810 µg/L)
NOECAdult Mortality : 270 µg/L


The recoveries in the fresh media were in the range of 73 to 86 % of the nominal values. In the old media (after 48 h or 72 h) the recoveries decreased to values in the range of 38 to 60 %. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame between the refreshments of the test solutions. The adsorption of the test item to the glassware was determined at the concentration level 30 µg/L on day 21 to be < LOD, which corresponds to < 0.0833 % of the nominal concentration. The limited concentration decrease between fresh and old media is therefore most likely caused by thermodynamically more favourable redistribution of the sorbed fraction resulting in an additional sorption to suspended matter and DOC. The results of the chemical analyses show that the test organisms were fully exposed to the test substance during the test. Therefore, all effect values given are based on the nominal test item concentrations.

Executive summary:

Two long-term daphnia studies are available for:

New Name: Fatty acids, C18 unsat, reaction products with diethylenetriamine (CAS no 1226892-43-8)

Old Name: Tall oil diethylenetriamine imidazoline (CAS no 68442-97-7)

Abbreviation: AAI-DETA

 

Both studies are performed to evaluate the long-term toxicity to aquatic invertebrates but follow two different approaches.

AAI-DETA is a multicomponent mixture (UVCB) of cationic surface-active constituents with different water solubilities. The fate of cationic surfactants in general deviates from standard chemicals. These substances are therefore considered as difficult substances for which the results of standard guideline studies are very difficult to interpret when considering them in a standard way. The reasons are the intrinsic properties like the relatively low water solubility and strong sorption to equipment and organisms. Classical ecotoxicity testing with these substances using reconstituted water often leads to test results which are poorly reproducible and are associated with high uncertainty. In addition, because of the complex sorption mechanisms (van der Waals and Ionic mechanisms) the actual dissolved exposure concentration cannot reliably be estimated.

The two available long-term tests where therefore performed following two different approaches.

 

One test which is focused on determining the intrinsic toxicity of AAI-DETA (for C&L purposes) is performed according to the Water Accommodate Fraction (WAF) approach as described in “OECD guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (No. 23 Feb. 2019) with a daily refreshment of the test solutions. The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration.

 

The other test which is more suited to derive a realistic risk ratio for the aquatic compartment is performed according to the PECaquatic bulk/PNECaquatic bulk approach as described in ECETOC Technical Report “Environmental Risk Assessment of difficult substances” (TR 88, 2003) with a three times a week refreshment of the test solutions. The so called “Bulk approach” is used in the environmental risk assessment to cope with the earlier mentioned lack of realistic PEC estimation. Instead of using the dissolved PECwater, the Bulk concentration (dissolved + sorbed) in water is used. This bulk approach requires a PNECwater, bulk that means that testing has to use river water which contains dissolved organic carbon and suspended organic and inorganic matter, instead of reconstituted water.

Tests according to the bulk approach were thus performed because the partitioning of cationic surfactants to soil, sediment or suspended matter is rather complex which explains why there is no alternative Equilibrium Partitioning Method (EPM, di Toro, 2008) formula for these substances available yet. The use of the Bulk approach however elegantly bypasses this deficiency as it eliminates the EPM on the exposure and effect side.

 

Main difference between the two approaches lies in the preparation of the test solutions and how the results should be interpreted.

• For the preparation of the test solutions according to the WAF approach, all reasonable efforts were taken to produce a solution of all soluble components of the test item in test media. The test solutions were prepared daily, by gentle mixing the test item with test medium for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphoning. This procedure was followed for each renewal of the test solutions. Five WAFs were prepared and tested at nominal loading rates 5.12-12.8-32.0-80.0-200 µg/L (separation factor 2.5), corresponding to the time weighted mean measured test item concentrations were 1.5-4.1-8.62-22.2 and 47.8 µg/L. No undissolved or emulsified material was observed in the WAF solutions based on the Tyndall effect check. Only the WAFs for the coating phase on day -1 showed a positive Tyndall effect.
  The dissolved fraction in the WAF solutions were analytically verified via LC-MS/MS three times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7 and 14) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8, 15) in all WAFs and in the control. All ionizable compounds were fragmented, detected and summed up in a TIC (total ion count) signal.
  Since the test item is a multicomponent mixture (UVCB), the test solution is considered a water accommodated fraction (WAF). The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration. For tests with chemicals that are not completely dissolved, the actual exposure concentration cannot be quantified by analytical methods at the concentrations causing effects. The effect concentration should then be expressed based on the nominal concentrations or loading rate (for mixtures).

A fingerprint was performed with the highest loading rate and compared with the analytical standard of the test item prepared in acetonitrile. Both were verified via MS and evaluated by the software. The detected signals of the analytical standard and of the test item solution were compared. In the standard solution two main groups could be identified, around 349 m/z and 611 m/z. In the WAF solution these groups could not be identified. The signals were not different to the signals of the control sample. No additional groups could be identified.
The results of the WAF test are therefore presented based on nominal Test Loadings. The TWA results are also given despite the fact that per definition of the WAF, all terms related to concentration level should be given as loading rates because partly dissolved compounds and mixtures cannot be related to concentrations. The results based on nominal and on Time Weighted Average (TWA) measured concentrations are: EL10/EC10 for reproduction after 21 days is 133/34.3 µg/L. The EL50/EC50 for adult mortality after 21 days is 111/29.1 µg/L

Because it is known that cationic surfactants sorb strongly to algae (van Wijk et al., 2009) it should be noted that the daphnids in the long-term daphnia test were to large extent exposed to the algae bound fraction of AAI-DETA via ingestion of algae. Basing the dose response on only the dissolved fraction would represent an extreme worst-case.

• The test solutions for the Bulk approach were prepared by diluting a stable emulsion of 10 mg/L in test medium. In agreement with the bulk approach the test medium used was natural surface water. The following concentrations were prepared by diluting the stock solution in test medium: 10, 30, 90, 270, and 810 µg/L. Due to the use of non-standard test medium (natural river water) the results of bulk approach test are considered inadequate by regulators involved in C&L because they do not fulfill to the narrow criteria set to quantify the intrinsic toxicity. There is however a clear difference in the evaluation of a standard aquatic ecotoxicity test and an ecotoxicity test performed using the Bulk approach. In order to class a standard laboratory toxicity study valid, it is of particular importance that – besides information on test substance, test method/conditions and test organism used - suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations are based upon measured levels of the dissolved concentration.

For ecotoxicity tests performed using the bulk approach, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the dose-response relationship should thus be based on the sum of adsorbed and dissolved substance. Results from bulk-approach tests are therefore easier to interpret because nominal concentrations corrected for sorption to glassware can be used to quantify the dose. Because of the use of natural river water a bulk approach test is more environmentally realistic than the standard method and due to that considered to be a higher tier study.

 

Droge & Goss (2013) have shown that sorption of cationic surfactants to soil and sediment is mainly driven by electrostatic interaction and to a lesser extent by hydrophobic interaction. This means that both the suspended matter and dissolved organic carbon in surface water are the key surface water properties determining the bioavailability of the test item.

The natural surface water was therefore characterized in detail and selected to contain a realistic worst-case suspended matter concentration of 15±3.5 mg/L and ± 3.5 mg/L DOC(≈NPOC). It should be noted that this composition is in perfect alignment with the risk assessment method developed by ECHA, as the concentration of suspended matter in surface water is considered to be 15 mg/L in CHESAR III for risk assessment (see ECHA’s guidance R.16, v3.0, Feb 2016, p. 88).

 

Sorption to glassware was observed to be <0.08% which means that the nominal test concentrations can be used for the dose-reponse because the recoveries in the fresh media were in the range of ≥80 % of the nominal values. The difference between the observed recoveries and nominal concentrations is explained by the rapid sorption of AAI-DETA to the river water constituents. Based on the study result, the NOEC for reproduction, mortality, adult length and weight of the test substance for Daphnia magna were determined to be 270 µg/L.

 

The degree of mitigation of the long term toxicity to daphnia due to the use of natural river water can be calculated by taking the ratio of the results observed for the bulk approach test and the nominal test results observed for the WAF approach test.

The mitigation factor for the chronic effect (NOEC/EC₁₀) to algae is 270/133 is 2.03

 

• Droge, S.T.J. and Goss, K.W. 2013. Development and Evaluation of a New Sorption Model for Organic Cations in Soil: Contributions from Organic Matter and Clay Minerals. Environmental Science and Technology, 47:14233-14241.


• Di Toro, D 2008 Bioavailability of chemicals in Sediments and soils: toxicological and chemical interactions. SERDP/ESTCP Bioavailability workshop


• van Wijk, D., Gyimesi-van den Bos, M., Garttener-Arends, I., Geurts, M., Kamstra, J., Thomas, P., 2009. Bioavailability and detoxification of cationics, I. Algal toxicity of trimethylammonium salts in the presence of suspended matter and humic acid. Chemosphere 75 (3), 303–309.

 

 

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2021-01-25 to 2021-02-17, with the definitive exposure phase from 2020-01-27 to 2020-02-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item Fatty acids, C18 unsat, reaction products with
diethylenetriamine

Commercial name ARMOHIB Cl

Batch number 1865159

CAS No. 1226892-43-8

Purity (certified) 100% UVCB

Appearance Liquid, amber, green

Water Solubility Critical micelle concentration (CMC): 99 mg/L (pH 7, 23 °C)

Stability under test conditions Not specified

Expiry date 2022-07-08

Recommended storage
Keep container tightly closed in a dry and well ventilated place, under nitrogen, room temperature, protected from light.

Analytical monitoring:

yes

Remarks:

via LC-MS/MS

Details on sampling:

Determination of the test item
All loading rates and the control were analyzed via LC-MS/MS at 3 intervals during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours) as well as in the old media at the end of an exposure-renewal interval (24 hours).
The samples were analyzed under GLP with an LC-MS/MS method which has been implemented under non-GLP and documented finally in the GLP raw data. The LC-MS/MS method was validated.
The old media samples were centrifuged to separate the algae from the water phase. Algae and water were analyzed separately to determine the truly dissolved test item fraction and the fraction adsorbed to algae. The centrifuge tubes were rinsed with the appropriate test solutions to minimize adsorption to the walls of the centrifuge tubes.

Sampling for the analytical monitoring
At the start of the exposure-renewal intervals, samples of the fresh media were taken after preparation of all loading rates and analyzed.
At the end of the exposure-renewal intervals (24 hours), samples of the old media were taken from the test vessels.

Vehicle:

no

Details on test solutions:

Water Accommodated Fraction
Water accommodated fractions (WAFs) were prepared because the test item is an UVCB substance with compounds of different water solubility. This procedure is in accordance with the OECD guidance document No. 23 (2019).
Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

Preparation of the water accommodated fractions
Five water accommodated fractions (WAFs) were prepared 24 ± 2 hours before the start of the exposure and each renewal day with nominal loading rates of the test item in the range of 5.12 - 12.8 - 32.0 - 80.0 - 200 µg test item/L, set up in a geometric series with a factor of 2.5. Five WAFs were prepared 48 ± 1 hour before the start of the exposure for the coating of the test vessels on day -1.
The procedure according to ASTM D6081 (2019) as described below was carried out for each exposure-renewal interval in the same way to achieve a constant quality during the course of the study.
Each water accommodated fraction (WAF) was prepared separate. For each loading rate an appropriate amount of the stock solution containing the test item in methanol was placed on a glass slide. The methanol was evaporated. The glass slide with the test item was inserted in a glass flask with an appropriate amount of dilution water. The dispersions were stirred with a magnetic stirrer for 24 ± 1 hour (1100 rpm, room temperature). After a separation phase of 1 hour, the aqueous phase of the WAF was removed by siphoning (from the approximate middle of the glass flask). The first 25 mL were discarded.
The WAFs were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). No presence of undissolved test item during the exposure phase was observed. The resulting water accommodated fractions (WAF) were used in the test.
For the preparation of the water accommodated fractions a stirring phase of 24 hours was found to be suitable. Longer stirring phases led to a decrease of the loading in the WAFs (< LCL). Therefore, a stirring phase of 24 hours was chosen for the definitive test.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
The test species was Daphnia magna STRAUS, obtained from continuous laboratory cultures. The daphnids of the laboratory culture originated from clone 5.

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline and is bred at the test facility.

The Daphnia magna culture has shown to meet the validity criteria for the reproduction test of a mean of ≥ 60 living juveniles per survived parent animal when cultured under conditions described.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2°C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx.

Culture medium
Elendt M4, according to OECD 211, Annex 2, and OECD 202, Annex 3, is used.

 
Feeding of the culture stocks
The daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae are cultured at the test facility.

Test type:

semi-static

Water media type:

freshwater

Remarks:

Medium Elendt M4

Limit test:

no

Total exposure duration:

21 d

Hardness:

See section "Any other information on results incl. tables".

Test temperature:

The incubator temperature was in the range of 19 to 21°C.
For more details see section "Any other information on results incl. tables".

pH:

See section "Any other information on results incl. tables".

Dissolved oxygen:

See section "Any other information on results incl. tables".

Nominal and measured concentrations:

Five water accommodated fractions (WAFs) were prepared 24 ± 2 hours before the start of the exposure and each renewal day with nominal loading rates of the test item in the range of 5.12 - 12.8 - 32.0 - 80.0 - 200 µg test item/L, set up in a geometric series with a factor of 2.5.

The test item concentrations of Fatty acids, C18 UNSAT, REACTION PRODUCTS WITH DIETHYLENETRIAMINE were analytically verified via LC-MS/MS three times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7 and 14) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8, 15) in all WAFs and in the control. The analytical results are presented in section "Any other information on results incl. tables".

Details on test conditions:

The definitive test was performed under semi-static conditions with a daily renewal of the test solutions and a coating phase of the test vessels. The stability of the test item over an exposure interval was confirmed in a non-GLP stability test.

Test loading
The loading rates are based on the results of a preliminary range finding test (non-GLP, open system with headspace).

Control 10 replicates of dilution water without test item

Test method
The study was performed under semi-static conditions with a daily renewal of the test solutions and a coating phase of the test vessels.

Test duration 21 days

Test vessels and pre-treatment
Glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity, loosely covered with watch glasses were used.
A coating phase (saturation of the test container) was carried out. The test containers were pre-treated with the appropriate test solution for at least 12 hours under test conditions. Before the start of the exposure and each renewal, the test container were emptied and refilled with freshly prepared test solutions.

Test volume 50 mL

Test medium Same composition as the culture medium

Number of daphnids and replicates
10 daphnids and 10 replicates were used for all WAFs and the control. 1 daphnid was held individually per replicate.

 
Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for exposure the study. To achieve this, juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within this period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization Acclimatization was not necessary, because the composition of the dilution water was equivalent to the culture medium.

Application 50 g test solution per replicate were weighed into the test vessels. This corresponds to 50 mL. The daphnids were transferred in a small amount of dilution water or test solution by pipette.

Renewal of the test solutions
The test solutions and the control were renewed daily. For this purpose, the freshly prepared test solutions were filled into a second set of test vessels and the daphnids were transferred by pipette (see ‘Application’).

Feeding
Daily feeding with unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, max. 0.2 mg C / daphnia and day

Test temperature (target)
The temperature should be in the range of 18 – 22 °C and should not vary by more than 2 °C.

Light intensity (target) Max. 1500 lx

Photoperiod 16/8 hours light/dark cycle

Aeration Test vessels were not aerated during the test.

Mortality of parental Daphnids
The number of parental daphnids, which were immobilised or dying during the test and the dates of death were observed and recorded once a day at least at the same time as the number of offspring was counted. Dead specimens were removed as soon as they were detected. Accidental mortality did not occur.

 
Offspring
Test vessels were checked daily for first appearance of juveniles. The number of neonates (alive and dead progeny) was counted and the dates of release were recorded once a day from the first day of production of neonates until the end of the exposure. The neonates (alive and dead) were removed after counting and before addition of algae to prevent them from consuming food intended for the adults. The number of aborted eggs or dead offspring and the dates of observation were recorded. At the end of the test (21 days), the total number of living offspring was assessed.

Additional observations / Condition of parental

Abnormalities (e.g. swimming behaviour, number of males and winter eggs)
Condition of parental were observed and recorded on each day of
daphnids observation.

Total body length / Mean dry At the end of the test, the total length excluding the anal spine of
weight of parental daphnids each survived parental daphnid and the mean dry weight of the survived parental daphnids of all WAFs and the control were determined. As stated in the OECD protocol, this may be useful additional information but should not be used for determination of a NOEL.

Additional endpoints
The time to first brood and the number and size of first brood per animal were reported, but not used for endpoint calculations.

Water Quality Parameters

Measurements
The water quality parameters (i.e. pH-values, dissolved oxygen concentration, total hardness and water temperature) were measured once within 7 days, in the fresh media at the start of an exposure-renewal interval (0 hours) as well as in the old media at the end of an exposure-renewal interval (24 hours), in one replicate of the control and the highest loading rate of the test item with a living parent animal.
The water quality parameters in the fresh media were measured in an additional replicate without daphnids of the highest loading rate of the test item and the control. At the end of the exposure-renewal interval, the water quality parameters of the old media were measured in a test vessel of the highest loading rate of the test item and the control, which contains daphnids and food algae.
The incubator temperature (measurement in air with a thermo-hygrograph) was recorded throughout the period of the test.



 

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

4.1 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

12.8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

8.62 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

32 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

other: LC100

Effect conc.:

47.8 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

other: LL100

Effect conc.:

200 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

29.1 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

LL50

Effect conc.:

111 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

other: LC20

Effect conc.:

14.7 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

other: LL20

Effect conc.:

49.4 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

22.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

80 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

47.8 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

200 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

36.8 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

146 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

34.3 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

133 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Biological Data

Reproductive Output

The number of juveniles in all replicates of the control and the test groups was counted and recorded every day.

The average number of living juveniles at the end of the test after 21 days per introduced parental female daphnid was 116 in the control group and thus the validity criterion for the reproduction of a mean of ≥ 60 living offspring per survived parent animal in the control was met.

Accidental and inadvertent mortality were not observed. Since the Cochran-Armitage trend test revealed a significant increasing trend (p ≤ 0.05) in adult mortality, the evaluation was done based on the cumulative offspring per introduced (female) parent.

A statistically significant reduction of the reproductive output in comparison to the reproductive output in the control was determined at the nominal loading rate 200 µg/L, corresponding to the time weighted mean measured concentration 47.8 µg/L. Therefore, the LOEL was determined to be 47.8 µg/L and the NOEL 22.2 µg/L for the cumulative offspring per introduced parent.
The determination of the EC/EL10/50-value and its 95% confidence limits was carried out by sigmoidal dose response regression. The EL10 for the test item was 133 µg/L and the EL50 146 µg/L per introduced parent. The EL10 for the test item was 34.3 µg/L and the EL50 36.8 µg/L per introduced parent.

Adult Mortality

Accidental and inadvertent mortality as defined in the OECD test guideline were not observed. In the control no mortality of the introduced adults was observed.
The test item induced adult mortality of 100% after 21 days in the nominal loading rate 200 µg/L, 20%, 30% and 10% adult mortality in the loading rate 80.0µg/L, 32.0 µg/l and 12.8 µg/L. No mortality was observed at the nominal loading rate 5.12 µg/L. The Cochran-Armitage test procedure revealed a significant increasing trend (p ≤ 0.05) in adult mortality. A statistically significant dose-response was detected. The determination of the LC/LL50-value was carried out by probit analysis. The confidence limits were estimated from the observed data. The LL50 was 111 µg/L (confidence limits: 80.0 – 200 µg/L). The LOELAdult mortality was 32.0 µg/L. The NOELAdult mortality was 12.8 µg/L. The LC50 was 29.1 µg/L (confidence limits: 22.2 – 47.8 µg/L). The LOECAdult mortality was 8.62 µg/L. The NOECAdult mortality was 4.10 µg/L.

Intrinsic Rates of Natural Increase (IR)

The intrinsic rate of population increase is a measure of population growth which integrates reproductive output and age-specific mortality. In steady state populations it will be zero. For growing populations it will be positive and for shrinking populations it will be negative.

First Appearance of living Juveniles

The first appearance of living juveniles was observed between days 7 and 9 at the introduced parental daphnids of the control and the nominal loading rates of 5.12 to 80.0 µg/L of the test item.

Analytical Results

The test item concentrations of Fatty acids, C18 UNSAT, REACTION PRODUCTS WITH DIETHYLENETRIAMINE were analytically verified via LC-MS/MS three times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7 and 14) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8, 15) in all WAFs and in the control.

Water Quality Parameters

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits.The incubator temperature was in the range of 19 to 21°C.

Validity Criteria

• Mortality of the parent animal in the control group should not exceed 20% at the end of the test. No parent animal in the control group died. Actual mortality of the parent animals in the control group was 0%.

• The mean number of living offspring produced per survived parent animal at the end of the test was 116 in the control group (required: at least 60 juveniles per survived parental daphnid).

Observations at Test and Control Groups
Stillborn Juveniles and Aborted Eggs

No aborted eggs were observed in all loading rates and the control during the exposure period of 21 days. In all loading rates and the control no stillborn juveniles were observed.

Growth (Total Body Length and Mean Dry Weight) of the Survived Parental
Daphnids

At the end of the test the mean values of the body length (excluding the anal spine) of the survived parental female daphnids in the tested loading rates of 5.12 to 80.0 µg/L were 4.5 mm per daphnid and 4.5 mm per female daphnid in the control group.
On day 21 the mean dry weight of the survived parental daphnids was in the range of 0.46 to 0.79 mg per daphnid in the WAF loading rates of 5.12 to 80.0 µg/L and 0.76 mg per daphnid in the control.

Presence of Males

No males were observed in the control or in any of the test groups during the test.

Occurrence of Ephippia (Winter Eggs)

No ephippia were observed in the control or in the test groups during the test.

Reported statistics and error estimates:

All calculations are based on the control and the nominal loading rates 5.12 – 12.8 – 32.0 – 80.0 - 200 µg/L and the corresponding time weighted mean measured test item concentrations.

Calculation of the time weighted mean concentration

Cw = time-weighted mean concentration
n = number of sampling periods
Cnew,i = concentration of the fresh test solution of period i
Cold,i = concentration of the old solution of period i
wi = time ti – ti-1, the number of hours or days in the ith interval
between measurements of concentration

Statistical evaluation
The NOEL/NOEC and LOEL/LOEC for the reproduction and the adult mortality were determined with the software ToxRat Professional as specified below. Significant differences of reproduction were determined in comparison to the control using statistical standard procedures as normality test (Shapiro-Wilk’s test), variance homogeneity test (Levene’s test), and a step-down Jonckheere-Terpstra test for reproduction.
The step-down Cochran-Armitage test was used for adult mortality. P values of the normality and the variance homogeneity test were 0.01.
For the effects on reproduction, the juveniles per introduced parent at the start of the test excluding accidental and inadvertent mortality (reproductive output) were counted. Since no accidental or inadvertent mortality was observed, no parental daphnids were excluded from the evaluation of the reproductive output.
The coefficients of variation around the mean number of living offspring produced per introduced parent and per survived parent in the control and the test groups were evaluated.

Determination of the EC/ELx- / LC/LLx-values
The EC/EL10 -, EC/EL50 – values for the reproductive output were calculated by sigmoidal dose response regression with the software GraphPad Prism.
LC/LL20-, LC/LL50- values for the adult mortality were calculated by asymmetric dose response regression with the software GraphPad Prism.

The confidence limits of the EC/EL10 -, EC/EL50 -, LC/LL20-, LC/LL50- values and the LC/LL100-value for the adult mortality were empirically derived from the observed data.

Calculation of the The intrinsic rate of natural increase (IR) was calculated for the
intrinsic rate survived parental daphnids with the following EULER-LOTKA-equation with the software ToxRat Professional.
Sum(e^( -A * IR)) * Sx * Jx = 1
A = Age
IR = Intrinsic rate of natural increase 
Sx = Survival rate on day x
Jx = Reproduction rate on day x

The intrinsic rate of natural increase is a measure of population growth which integrates reproductive output and age-specific mortality. In steady state populations, it will be zero. For growing populations, it will be positive and for shrinking populations, it will be negative. Clearly, the latter is not sustainable and ultimately will lead to extinction.

Software

All data are computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- ToxRat Professional, Version 3.3.0, TOXRAT SOLUTIONS GMBH
- Excel, MICROSOFT CORPORATION
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

 

Effects on Reproduction for all Introduced Parents
(based on the nominal loadings of the water accommodated fractions of the test item [mg/L])

Nominal loading rate	Mean number of offspring
of the test item	per introduced parental daphnid
[µg/L]	Mean	SD	CV
200	0	0	0
80.0	114	30.0	26.3
32.0	102	16.3	16.0
12.8	110	16.3	14.9
5.12	120	10.7	8.9
Control	116	10.8	9.4

 

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure
(n = 10)

Nominal loading rate	Adult Mortality [%]
of the test item
[µg/L]	7 days	14 days	21 days
200	100	100	100
80.0	0	10	20
32.0	0	10	30
12.8	0	0	10
5.12	0	0	0
Control	0	0	0

 

Intrinsic Rates of Natural Increase

Nominal loading rate	Mean IR	CV
of the test item
[µg/L]	MV ± SD	[%]
200	n.a.	n.a.
80.0	0.413 ± 0.0188	4.5
32.0	0.409 ± 0.0241	5.9
12.8	0.411 ± 0.0114	2.8
5.12	0.421 ± 0.0173	4.1
Control	0.396 ± 0.0146	3.7

 

First Appearance of Living Juveniles in the Individual Groups

Nominal loading rate	Day of first appearance of living juveniles at the introduced parental daphnids in replicate no.										First
of the test item											appearance
[µg/L]	1	2	3	4	5	6	7	8	9	10	(mean day)
200	--	--	--	--	--	--	--	--	--	--	--
80.0	7	7	7	7	7	7	7	7	8	7	7.1
32.0	7	7	7	7	9	7	7	7	7	7	7.2
12.8	7	7	7	7	7	7	7	7	7	7	7.0
5.12	7	7	7	7	7	7	7	7	7	7	7.0
Control	7	7	7	7	7	7	7	8	7	7	7.1

= not applicable, since no living juveniles occurred

 

Measured Concentrations of the Test Item during the Definitive Test
(Days 0, 1, 7, 8, 14 and 15)

Sampling date		Day 0		Day 1		Day 7		Day 8
		Fresh medium		Old medium		Fresh medium		Old medium
		0 hours		24 hours		0 hours		24 hours
Nominal		Fatty acids, C18 unsat, reaction products with diethylenetriamine
loading rates
of the test item
[µg/L]		Meas.	%	Meas.	%	Meas.	%	Meas.	%
		conc.		conc.		conc.		conc.
		[µg/L]		[µg/L]		[µg/L]		[µg/L]
200		156	78	6.92	3	-
80.0		62.3	78	< LOQ		69.6	87	7.68	10
32.0		22.4	70	< LOQ		18.7	59	3.16	10
12.8		7.64	60	< LOQ		9.99	78	< LOQ
5.12		2.20*	43*	< LOQ		< LOQ		< LOQ
Control		< LOQ		< LOQ		< LOQ		< LOQ
.	Day 14			Day 15
	Fresh medium			Old medium
	0 hours			24 hours
Nominal	Fatty acids, C18 unsat, reaction products with diethylenetriamine
loading rates
of the test item
[µg/L]	Meas.		%	Meas.	%
	conc.			conc.
	[µg/L]			[µg/L]
200	-
80.0	70.6		88	3.42	4
32.0	29.4		92	< LOQ
12.8	8.54		67	< LOQ
5.12	2.64*		52*	< LOQ
Control	< LOQ			< LOQ

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration of the test item
LOQ = limit of quantification (3.00 µg/L of the test item)
- = not determined, due to 100% mortality
* = < LOQ but > 70% of the LOQ

 

Measured Concentrations of the Test Item during the Definitive Test (Algae Adsorption)
(Days 1, 8 and 15)

Sampling date	Day 1		Day 8		Day 15
	Old medium		Old medium		Old medium
	24 hours		24 hours		24 hours
Nominal	Fatty acids, C18 unsat, reaction products with diethylenetriamine
loading rates
of the test item
[µg/L]	Meas.	%	Meas.	%	Meas.	%
	conc.		conc.		conc.
	[µg/L]		[µg/L]		[µg/L]
200	44.9	22	-		-
80.0	12.2	15	18.6	23	10.6	13
32.0	< LOQ		4.05	13	3.04	9
12.8	< LOQ		< LOQ		< LOQ
5.12	< LOQ		< LOQ		< LOQ
Control	< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration of the test item
LOQ = limit of quantification (3.00 µg/L of the test item)
- = not determined, due to 100% mortality

 

Endpoints for Reproduction and Mortality
(based on the nominal loading rates of the test item and the time weighted mean measured test item concentrations)

the time weighted mean measured concentrations

Effect values	Fatty acids, C18 unsat, reaction products with diethylenetriamine
	based on	based on
	the nominal loading rates
	[µg/L]	[µg/L]
EL/EC10 Reproduction (introduced)	133 (Cl: 12.8 – 200)	34.3 (Cl: 4.10 – 47.8)
EL/EC50 Reproduction (introduced)	146 (Cl: 80.0 – 200)	36.8 (CI: 22.2 – 47.8)
LOEL/LOEC Reproduction (introduced)	200	47.8
NOEL/NOEC Reproduction (introduced)	80.0	22.2
LL/LC20 Adult mortality after 21 days	49.4 (CI: 5.12 - 200)	14.7 (CI: 1.50 – 47.8)
LL/LC50 Adult mortality after 21 days	111 (CI: 80.0 - 200)	29.1 (CI: 22.2 – 47.8)
LL/LC100 Adult mortality after 21 days	200	47.8
LOEL/LOEC Adult mortality after 21 days	32.0	8.62
NOEL/NOEC Adult mortality after 21 days	12.8	4.10

 

Stillborn Juveniles and Aborted Eggs produced by the Survived Parental Daphnids
after 21 Days

 

Nominal	Number of		Total no.
loading rate
of the test item			å
[µg/L]	stillborn	aborted
	juveniles	eggs
200	0	0	0
80.0	0	0	0
32.0	0	0	0
12.8	0	0	0
5.12	0	0	0
Control	0	0	0

 

Total Body Length and Dry Weight of the Survived Parental Daphnids

Nominal	Total length of the survived parent animals [mm]										MV	N	Dry weight
loading rate													[mg]
of the test item	Replicate no.
[µg/L]	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
200	-	-	-	-	-	-	-	-	-	-	-	-	-	-
80.0	4.5	-	4.5	4.5	4.5	4.5	-	4.5	4.5	4.5	4.5	8	4.1	0.51
32.0	4.5	4.5	4.5	-	-	-	4.5	4.5	4.5	4.5	4.5	7	3.2	0.46
12.8	4.5	4.5	4.5	4.5	4.5	4.5	4.5	4.5	4.5	-	4.5	9	6.9	0.77
5.12	4.5	4.5	4.5	4.5	4.5	4.5	4.5	4.5	4.5	4.5	4.5	10	7.9	0.79
Control	4.5	4.5	4.5	4.5	4.5	4.5	4.5	4.5	4.5	4.5	4.5	10	7.6	0.76

N = number of the survived parental female daphnids
- = not applicable, due to the mortality of the parental daphnid(s)

 

Observations in the Control

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	9	1	-	19	-	-	28	-	-	29	-	-	31	-	-	117
2	-	-	-	-	-	-	12	-	-	16	-	-	28	-	-	31	-	-	29	-	-	116
3	-	-	-	-	-	-	9	2	-	14	-	-	27	-	-	34	-	-	29	-	-	115
4	-	-	-	-	-	-	8	-	-	17	-	-	33	-	-	35	-	-	30	-	-	123
5	-	-	-	-	-	-	11	-	-	21	-	-	18	-	-	31	-	-	20	-	-	101
6	-	-	-	-	-	-	12	-	-	16	-	-	27	-	-	27	-	-	31	-	-	113
7	-	-	-	-	-	-	15	-	-	19	-	-	27	-	-	32	-	-	40	-	-	133
8	-	-	-	-	-	-	-	8	-	21	-	-	25	-	-	27	-	-	23	-	-	104
9	-	-	-	-	-	-	13	-	-	9	-	-	23	-	-	32	-	-	53	-	-	130
10	-	-	-	-	-	-	13	-	-	14	-	-	24	-	-	25	-	-	28	-	-	104
Based on all parental daphnids inserted at the start of the exposure:																						1156
Based on parental daphnids survived until the end of the test:																						1156
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Observations in the Nominal Loading Rate of 5.12 µg/L

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	17	-	-	21	-	-	26	-	-	30	-	-	31	-	-	125
2	-	-	-	-	-	-	15	-	-	19	-	-	25	-	-	34	-	-	30	-	-	123
3	-	-	-	-	-	-	15	-	-	21	-	-	26	-	-	32	-	-	25	-	-	119
4	-	-	-	-	-	-	11	1	-	22	-	-	31	-	-	32	-	-	21	-	-	118
5	-	-	-	-	-	-	14	-	-	13	-	-	25	-	-	28	-	-	22	-	-	102
6	-	-	-	-	-	-	16	-	-	23	-	-	29	-	-	32	-	-	34	-	-	134
7	-	-	-	-	-	-	17	-	-	21	-	-	29	-	-	25	-	-	27	-	-	119
8	-	-	-	-	-	-	12	-	-	12	-	-	25	-	-	32	-	-	33	-	-	114
9	-	-	-	-	-	-	13	-	-	18	-	-	25	-	-	32	-	-	23	-	-	111
10	-	-	-	-	-	-	19	-	-	25	-	-	30	-	-	35	-	-	30	-	-	139
Based on all parental daphnids inserted at the start of the exposure:																						1204
Based on parental daphnids survived until the end of the test:																						1204
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Observations in the Nominal Loading Rate of 12.8 µg/L

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	13	-	-	21	-	-	26	-	-	11	-	-	23	-	-	94
2	-	-	-	-	-	-	12	-	-	17	-	-	27	-	-	30	-	-	23	-	-	109
3	-	-	-	-	-	-	13	-	-	17	-	19	4	-	5	20	-	24	-	-	-	102
4	-	-	-	-	-	-	14	-	-	12	-	22	-	-	29	-	-	35	-	-	28	140
5	-	-	-	-	-	-	13	-	9	7	-	22	-	-	32	-	-	20	-	-	21	124
6	-	-	-	-	-	-	18	-	-	18	-	-	25	-	-	29	-	-	26	-	-	116
7	-	-	-	-	-	-	16	-	-	21	-	-	24	-	-	29	-	-	26	-	-	116
8	-	-	-	-	-	-	13	-	-	13	-	-	23	-	-	28	-	-	34	-	-	111
9	-	-	-	-	-	-	13	-	-	16	-	-	21	-	-	30M						80
10	-	-	-	-	-	-	12	-	-	13	-	-	29	-	-	28	-	-	25	-	-	107
Based on all parental daphnids inserted at the start of the exposure:																						1099
Based on parental daphnids survived until the end of the test:																						1019
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	1	1	1	1	1	1
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	10	10	10	10	10	10	10

Observations in the Nominal Loading Rate of 32.0 µg/L

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	16	1	-	17	-	-	23	-	-	29	-	-	21	-	-	107
2	-	-	-	-	-	-	11	-	-	16	-	-	28	-	-	30	-	-	28	-	-	113
3	-	-	-	-	-	-	19	-	-	19	-	-	27	-	-	28	-	-	33	-	-	126
4	-	-	-	-	-	-	18	-	-	24	-	-	34	-	-	28	-	M				104
5	-	-	-	-	-	-	-	-	11	-	18	-	-	34	-	-	33M					96
6	-	-	-	-	-	-	13	-	-	23	-	28	-	M								64
7	-	-	-	-	-	-	12	-	-	15	-	-	26	-	-	28	-	-	26	-	-	107
8	-	-	-	-	-	-	11	-	8	-	-	17	-	-	23	-	-	25	-	-	19	103
9	-	-	-	-	-	-	16	-	-	10	-	-	21	1	-	25	-	-	18	-	-	91
10	-	-	-	-	-	-	14	-	-	18	-	-	20	-	-	24	-	-	-	33	-	109
Based on all parental daphnids inserted at the start of the exposure:																						1020
Based on parental daphnids survived until the end of the test:																						756
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	0	1	1	1	2	3	3	3	3	3
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	10	10	10	20	30	30	30	30	30

Observations in the Nominal Loading Rate of 80.0 µg/L

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	13	-	-	20	-	2	29	-	-	28	-	-	27	-	-	119
2	-	-	-	-	-	-	14	-	-	19	-	1	27	-	-	31	-	-	19	M		111
3	-	-	-	-	-	-	21	-	-	9	-	29	-	-	39	-	-	21	-	-	14	133
4	-	-	-	-	-	-	16	-	-	21	-	23	-	-	31	-	-	21	-	-	17	129
5	-	-	-	-	-	-	12	-	-	12	-	27	-	-	30	-	-	37	-	-	18	136
6	-	-	-	-	-	-	21	-	-	13	-	28	-	-	39	-	-	27	-	-	-	128
7	-	-	-	-	-	-	11	1	-	21	-1	-1	M									33
8	-	-	-	-	-	-	12	1	-	13	-	28	-	-	6	31	-	28	-	-	-	119
9	-	-	-	-	-	-	-	8	1	-	19	-	-	33	-	-	31	-	-	33	-	125
10	-	-	-	-	-	-	11	1	-	17	-	8	18	-	-	22	-	29	-	-	-	106
Based on all parental daphnids inserted at the start of the exposure:																						1139
Based on parental daphnids survived until the end of the test:																						995
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	1	1	1	1	1	1	1	2	2	2
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	10	10	10	10	10	10	10	20	20	20

Observations in the Nominal Loading Rate of 200 µg/L

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	M																		0
2	-	-	-	-1	M																	0
3	-	-	-	M																		0
4	-	-	-	M																		0
5	-	-	-	M																		0
6	-	-	-	-	-1	M																0
7	-	-	-	-1	M																	0
8	-	-	-	-1	M																	0
9	-	-	-	M																		0
10	-	-	-	-1	M																	0
Based on all parental daphnids inserted at the start of the exposure:																						0
Based on parental daphnids survived until the end of the test:																						0
Mortality of Parental (N)	0	0	0	5	90	10																10
Mortality of Parental [%]	0	0	0	50	90	100																100

- = no juveniles observed
M = dead parental daphnid
1 = pale parental daphnid

 

Water Quality Parameters

Comparison of water quality parameters in fresh and old medium once within 7 days.

pH-Values

Test group	fresh	old	fresh	old	fresh	old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
Highest loading rate	7.59	7.88	7.45	7.51	7.53	7.89
Control	7.82	7.86	7.43	7.80	7.56	7.94

The pH-values should be in the range of 6 – 9. Significant deviations ≥ 1.5 units between the initial and final pH-values were not observed.

Dissolved Oxygen Concentration [mg/L]

Test group	fresh	old	fresh	old	fresh	old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
Highest loading rate	8.75	6.41	7.90	6.54	7.84	6.59
Control	8.27	5.79	7.72	6.72	7.64	6.69

The dissolved oxygen concentration was above 3 mg/L.

Total Hardness [mg/L] as CaCO3

Test group	fresh	old	fresh	old	fresh	old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
Highest loading rate	274	271	262	263	241	253
Control	255	251	265	255	294	245

The total hardness was above 140 mg/L as CaCO3.

Temperature [°C]

Test group	fresh	old	fresh	old	fresh	old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
Highest loading rate	21.0	21.5	21.7	21.8	21.4	20.6
Control	21.1	21.4	21.4	21.7	21.3	20.5

The temperature was within the range of 20 ± 2 °C.

 

MS Parameter

Parent Ion (Da)	Daughter Ion (Da)	Cone voltage (V)	Collision energy (eV)
104.08	87.09	20	8
346.28	97.15	66	48
	303.25		28
348.30	97.90	60	48
	305.26		28
350.31	97.09	68	48
	307.28		30
364.29	97.40	52	48
	304.22		22
366.31	306.24	48	22
	348.39		16
368.32	308.25	48	24
	350.46		16
606.50	97.34	74	68
	303.42		40
608.51	97.34	72	72
	302.96		42
610.53	97.27	74	78
	305.44		42
612.54	97.27	80	78
	305.05		42
614.56	97.27	80	80
	307.58		42
624.57	97.40	72	76
	319.48		44
626.59	97.93	74	52
	321.50		44
628.54	98.00	72	54
	321.69		42
630.55	98.00	68	54
	306.02		40
632.57	307.97	70	44

 

Dilution Steps

Nominal	Dilution	Sample	Final
concentration	Factor*	volume	volume
[µg/L]		[mL]	[mL]
200	25	0.08	1.0
80.0	10	0.2	1.0
32.0	4	0.5	1.0

*including factor 2

 

Dilution Steps (Algae Samples)

Nominal	Enrichment factor*	Sample	Final
concentration		volume	volume
[µg/L]		[mL]	[mL]
200	0.15	0.1	1.0
80.0	0.3	0.2	1.0

*including enrichment factor 1.5

Preparation of Fortified Samples

LOQ Level	Control	1	80
Stock solution	-	1000
[mg test item/L]		(Acetonitrile)
(Medium)
Spiking solution	-	0.300	24.0
[mg test item/L]		(Dilution medium)	(Dilution medium)
(Medium)
Replicates	2	5	5
Concentration of the LOQ	-	3.00	240
[µg test item/L]
Medium for preparation	Dilution medium
Volume of spiking solution [mL]	-	0.05	0.05
Volume of medium [mL]	10	9.95	9.95
Dilution factor	2	2	40
Dilution medium	Dilution medium
Sample volume [mL]	10^1)	10^1)	10^1)
			0.05^2)
Finale volume [mL]	10^1)	10^1)	10^1)
			1.0^2)

Dilution medium = Acetonitrile : daphnia dilution medium (50 . 509 containing 1% trifluoro acetic acid
1) First dilution step (dilution factor 2 included)

 

Recovery Rates of the Fortified Samples of the Test Item
Fortified concentrations*:
3.01 µg test item/L (1 x LOQ) and 240 µg test item/L (80 x LOQ)

Replicate	Fatty acids, C18 unsat, reaction products with diethylenetriamine
	1 x LOQ		80 x LOQ
	Meas. conc.	%	Meas. conc.	%
	[µg/L]		[µg/L]
1	3.29	110	229	95
2	3.28	109	251	104
3	3.31	110	265	110
4	3.36	112	248	103
5	3.18	106	260	108
Mean	3.28	109	251	104
SD ±	0.07		14
CV [%]	2.0		5.6

Meas. conc. = measured concentration, dilution factor taken into account
% = percent of the fortified concentration
* = weighing factor taken into account
SD = standard deviation
CV = coefficient of variation

 

Procedural Recovery

A procedural recovery (Quality Control) on 1x LOQ Level were freshly prepared on each day of analysis. They were treated in parallel to the test samples.

Measured Concentrations and Percent of Nominal Concentration of the Quality Control during the Definitive Test

Sampling	Day 0		Day 1		Day 7
date
Quality Control	Fatty acids, C18 unsat, reaction products with diethylenetriamine
[µg/L]	Meas.	%	Meas.	%	Meas.	%
	conc.		conc.		conc.
	[µg/L]		[µg/L]		[µg/L]
3.00	2.78	92	2.30	76	3.17	105
			3.40^1)	112^1)
Sampling	Day 8		Day 14		Day 15
date
Quality Control	Fatty acids, C18 unsat, reaction products with diethylenetriamine
[µg/L]	Meas.	%	Meas.	%	Meas.	%
	conc.		conc.		conc.
	[µg/L]		[µg/L]		[µg/L]
3.00	3.39	110	2.78	90	2.99	97
	2.65^2)	86^2)			2.58^2)	84^2)

Meas. conc. = measured concentration of the test item, dilution and weighing factors taken into account
% = percent of the nominal concentration
1) = reinjection (> 110%, but in the range of the mean recovery +/- 2x relative standard deviation of the 1xLOQ
in comparison to the method validation.
2) = sample set with algae adsorption samples

 

Preliminary Range Finding Test (non-GLP)

Solubility Test

Two WAF’s were prepared as described. After 24, 48 and 72 hours samples were taken as shown behind and analyzed via LC-MS.

Measured Concentrations of Fatty acids, C18 unsat, reaction products with diethylenetriamine during the Stability Test (non-GLP)
Analytical system: LC-MS

	Fatty acids, C18 unsat, reaction products with diethylenetriamine
	Measured concentration [µg/L]
Sampling date	24 hours	48 hours	72 hours
Nominal test item loading rate 1000 µg/L	392	329	272
Nominal test item loading rate 10 µg/L	< LCL	< LCL	< LCL

LCL = lowest calibration standard = 5 µg/L

 

Range Finding Test

A non-GLP preliminary range finding test was conducted at the test facility as a shortened reproduction test (at least until observation of the first brood in all test groups) under semi-static conditions with renewal of the test solutions every 2 – 3 days over a period of 8 days. A control and three water accommodated fractions (WAFs) with loadings levels of 50.0, 500 and 5000 µg test item/L were tested in the range finding test. The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle). Five daphnids, one daphnia per replicate, were exposed to each loading rate and the control.
The loading rates were analytically verified via LC-MS/MS in the fresh media (WAF, before distribution to the test vessels) at the start of the exposure and in the old media (test vessels containing daphnia and algae) at the end of an exposure-renewal interval.
For the preparation of the water accommodated fractions a stirring phase of 24 hours was found to be suitable. Longer stirring phases led to a decrease of the loading in the WAFs. Therefore, a stirring phase of 24 hours is chosen for the definitive test.

Immobilization and Offspring in the non-GLP Preliminary Range Finding Test after 8 Days
(n = 5, divided into 5 replicates with 1 daphnid each)

Nominal loading rates	Mortality [%] after 8 days						Number of offspring after 8 days per inserted parent						First appearance of living juveniles
of the WAF	Replicates						Replicates
[µg/L]	1	2	3	4	5	MV	1	2	3	4	5	MV	[d]
5000	100	100	100	100	100	100	0	0	0	0	0	0	-
500	100	100	100	100	100	100	0	0	0	0	0	0	-
50	0	0	0	0	0	0	12	18	12	10	11	13	8
Control	0	0	0	0	0	0	16	14	18	12	21	16	7 – 8

 

 

Measured Concentrations of the Test Item during the non-GLP Preliminary Range Finding Test Determination of the test item via LC-MS/MS

Sampling date	Fresh media, 0 hours,		Old media, 48 hours,
	study day 0		study day 2
Nominal loading rates	Fatty acids, C18 unsat, reaction products with diethylenetriamine
of the WAF
[µg/L]	Measured concentration^1)	%	Measured concentration^1)	%
	[µg/L]		[µg/L]
5000	4147	83	2047	41
500	401	80	47.1	9
50	6.09	12	< LCL
Control	< LCL		< LCL

Measured Concentrations of the Test Item during the non-GLP Preliminary Range Finding Test Determination of the test item via LC-MS/MS

Sampling date	Fresh media, 0 hours,		Old media, 72 hours,
	study day 2		study day 5
Nominal loading rates	Fatty acids, C18 unsat, reaction products with diethylenetriamine
of the WAF
[µg/L]	Measured concentration^1)	%	Measured concentration^1)	%
	[µg/L]		[µg/L]
5000	Not determined, due to 100% mortality of the parent animals in all replicates of the concentration level.
500	464	93	55.2	12
50	30.1	60	2.98	10
Control	< LCL		< LCL

1) = dilution factors taken into account
% = percent of the nominal concentration of the test item
LCL = lowest calibration level (1 µg/L of the test item)

 

MS-Fingerprints (non-GLP)

The peak distribution of the WAF was analyzed in fresh prepared medium in the highest test item concentration 200 µg/L. Additionally, the control sample of the main test was analysed. An analytical standard of the test item was prepared in acetonitrile and diluted to 160 µg/L with acetonitrile. The highest test item concentration was diluted factor 2 with acetonitrile containing 2% trifluoroacetic acid to avoid an inhomogeneous sample. The standard dilution and the test item dilution were analytical verified via MS and evaluated by the software. The detected signals of the analytical standard and of the test item solution were compared. In the standard solution two main groups could be identified, around 349 m/z and 611 m/z. In the WAF solution these groups could not be identified. The signals were not different to the signals of the control sample. No additional groups could be identified.

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

Conclusions

The effects on reproduction were evaluated based on the reproduction per female parent animal inserted at the start of the exposure. The Cochran-Armitage trend-test revealed a significant trend in mortality. Thus, according to OECD 211 it is required to report the effect values for the cumulative offspring per introduced parent provided these values are lower than in the cumulative offspring per survivor.
A summary of all endpoints based on the nominal loading rates of Fatty acids, C18 unsat, REACTION PRODUCTS WITH DIETHYLENETRIAMINE is given in the table below. The overall effect threshold for effects of the test item under the test conditions was 80.0 µg/L (NOEL) and 200 µg/L (LOEL) based on the nominal loading rates of the test item Fatty acids, C18 unsat, REACTION PRODUCTS WITH DIETHYLENETRIAMINE.
The overall effect threshold for effects of the test item under the test conditions was 47.8 µg/L (LOEC) and 22.2 µg/L (NOEC) based on the time weighted mean measured concentrations of the test item Fatty acids, C18 unsat, REACTION PRODUCTS WITH DIETHYLENETRIAMINE.

Executive summary:

A Daphnia magna reproduction test (semi-static, 21 d) with Fatty acids, C18 unsat, REACTION PRODUCTS WITH DIETHYLENETRIAMINE (AAI-DETA) was performed according to OECD 211 (2012) under GLP conditions.
Test species was Daphnia magna STRAUS. Ten daphnids, held individually, were used per WAF loading rate and control. At the start of the exposure the daphnids were less than 24 hours old. The study was carried out under semi-static conditions with a daily renewal of the test solutions. Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, the number of broods with living juveniles, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.
AAI-DETA is a UVCB substance with compounds of different water solubility. The aqueous media were prepared following OECD guidance document No. 23 (2019), which is specifically developed for aquatic toxicity testing of UVCBs, by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphon. This procedure was followed for each renewal of the test solutions. Five WAFs were prepared and tested at nominal loading rates 5.12 – 12.8 – 32.0 – 80.0 – 200 µg/L (separation factor 2.5), corresponding to the time weighted mean measured concentrations 1.50 – 4.10 – 8.62 – 22.2 – 47.8 µg/L.
The WAFs were checked for any undissolved or emulsified material by Tyndall effect throughout the exposure phase, which was negative. Only the WAFs for the coating phase on day -1 showed a positive Tyndall effect.
The AAI-DETA were analytically verified via LC-MS/MS three times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7 and 14) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8, 15) in all WAFs and in the control. All ionizable compounds were fragmented, detected and summed up in a TIC (total ion count) signal.
The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met.

The effects on reproduction were evaluated based on the reproduction per female parent animal introduced at the start of the exposure. The Cochran-Armitage trend-test revealed a significant trend in mortality. Thus, according to OECD 211 it is required to report the effect values for the cumulative offspring per introduced parent provided these values are lower than in the cumulative offspring per survivor. All reasonable efforts were taken to produce a solution of all soluble components of the test item in test media. Since the test item is a multicomponent mixture (UVCB), the test solution is considered a water accommodated fraction (WAF). The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration. For tests with chemicals that are not completely dissolved, the actual exposure concentration cannot be quantified by analytical methods at the concentrations causing effects. The effect concentration should then be expressed based on the nominal concentrations or loading rate (for mixtures). An MS fingerprint was done with the highest loading rate and compared with the analytical standard of the test item prepared in acetonitrile. Both were verified via MS and evaluated by the software. The detected signals of the analytical standard and of the test item solution were compared. In the standard solution two main groups could be identified, around 349 m/z and 611 m/z. In the WAF solution these groups could not be identified. The signals were not different to the signals of the control sample. No additional groups could be identified.
All evaluations are based on the nominal loading rates of Fatty acids, C18 unsat, REACTION PRODUCTS WITH DIETHYLENETRIAMINE. All calculations are based on the control and the nominal loading 5.12 - 12.8 - 32.0 - 80.0 - 200 µg/L and the corresponding time weighted mean measured test item concentrations.
A summary of all endpoints based on the nominal loading rates of Fatty acids, C18 unsat, REACTION PRODUCTS WITH DIETHYLENETRIAMINE is given in the table below.

 

Endpoints for Reproduction and Mortality
(based on the nominal loading rates of the test item and the time weighted mean measured test item concentrations)

Effect values	Fatty acids, C18 unsat, reaction products with diethylenetriamine
	based on	based on
	the nominal loading rates	the time weighted mean measured concentrations
	[µg/L]	[µg/L]
EL/EC10 Reproduction (introduced)	133 (Cl: 12.8 – 200)	34.3 (Cl: 4.10 – 47.8)
EL/EC50 Reproduction (introduced)	146 (Cl: 80.0 – 200)	36.8 (CI: 22.2 – 47.8)
LOEL/LOEC Reproduction (introduced)	200	47.8
NOEL/NOEC Reproduction (introduced)	80.0	22.2
LL/LC20 Adult mortality after 21 days	49.4 (CI: 5.12 - 200)	14.7 (CI: 1.50 – 47.8)
LL/LC50 Adult mortality after 21 days	111 (CI: 80.0 - 200)	29.1 (CI: 22.2 – 47.8)
LL/LC100 Adult mortality after 21 days	200	47.8
LOEL/LOEC Adult mortality after 21 days	32.0	8.62
NOEL/NOEC Adult mortality after 21 days	12.8	4.10

CI = confidence limits

Description of key information

Two long-term daphnia studies are available for: Fatty acids, C18 unsat, reaction products with diethylenetriamine (CAS no 1226892-43-8) Abbreviation: AAI-DETA

Both studies are performed to evaluate the long-term toxicity to aquatic invertebrates but follow two different approaches. One study which is performed focusing on the determation of the intrinsic toxicity of AAI-DETA (for C&L purposes) according to the Water Accommodate Fraction (WAF) approach as described in “OECD guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (No. 23 Feb. 2019) resulted in a 21d EL10 of 133 µg/L

The other study which is more suited to derive a realistic risk ratio for the aquatic compartment is performed according to the PECaquatic bulk/PNECaquatic bulk approach as described in ECETOC Technical Report “Environmental Risk Assessment of difficult substances” (TR 88, 2003) resulted in a 21 d EC10 for reproduction of 270 µg/L based on the nomimal test substance concentration.

Due to the use of riverwater in the Bulk approach test the toxicity may have been mitigated by reduction of the dissolved concentration due to sorption to river water constituents (suspended matter and DOC). The size of this mitigation factor can be calculated from the ratio between the Bulk approach EC10 and WAF approach EL10: 270/133 = 2

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Remarks:

133

Effect concentration:

133 µg/L

Additional information

The test item has been subjected to aquatic ecotoxicity tests following two different approaches. The WAF and Bulk approach.

Main difference between the WAF and Bulk appoach lies in the preparation of the test solutions and how the results should be interpreted.

• For the preparation of the test solutions according to the WAF approach, all reasonable efforts were taken to produce a solution of all soluble components of the test item in test media. The test solutions were prepared daily, by gentle mixing the test item with test medium for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphoning. This procedure was followed for each renewal of the test solutions. Five WAFs were prepared and tested at nominal loading rates 5.12-12.8-32.0-80.0-200 µg/L (separation factor 2.5), corresponding to the time weighted mean measured test item concentrations were 1.5-4.1-8.62-22.2 and 47.8 µg/L. No undissolved or emulsified material was observed in the WAF solutions based on the Tyndall effect check. Only the WAFs for the coating phase on day -1 showed a positive Tyndall effect.
  The dissolved fraction in the WAF solutions were analytically verified via LC-MS/MS three times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7 and 14) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8, 15) in all WAFs and in the control. All ionizable compounds were fragmented, detected and summed up in a TIC (total ion count) signal.
  Since the test item is a multicomponent mixture (UVCB), the test solution is considered a water accommodated fraction (WAF). The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration. For tests with chemicals that are not completely dissolved, the actual exposure concentration cannot be quantified by analytical methods at the concentrations causing effects. The effect concentration should then be expressed based on the nominal concentrations or loading rate (for mixtures).

A fingerprint was performed with the highest loading rate and compared with the analytical standard of the test item prepared in acetonitrile. Both were verified via MS and evaluated by the software. The detected signals of the analytical standard and of the test item solution were compared. In the standard solution two main groups could be identified, around 349 m/z and 611 m/z. In the WAF solution these groups could not be identified. The signals were not different to the signals of the control sample. No additional groups could be identified.
The results of the WAF test are therefore presented based on nominal Test Loadings. The TWA results are also given despite the fact that per definition of the WAF, all terms related to concentration level should be given as loading rates because partly dissolved compounds and mixtures cannot be related to concentrations. The results based on nominal and on Time Weighted Average (TWA) measured concentrations are: EL10/EC10 for reproduction after 21 days is 133/34.3 µg/L. The EL50/EC50 for adult mortality after 21 days is 111/29.1 µg/L

Because it is known that cationic surfactants sorb strongly to algae (van Wijk et al., 2009) it should be noted that the daphnids in the long-term daphnia test were to large extent exposed to the algae bound fraction of AAI-DETA via ingestion of algae.

• The test solutions for the Bulk approach were prepared by diluting a stable emulsion of 10 mg/L in test medium. In agreement with the bulk approach the test medium used was natural surface water. The following concentrations were prepared by diluting the stock solution in test medium: 10, 30, 90, 270, and 810 µg/L. Due to the use of non-standard test medium (natural river water) the results of bulk approach test are considered inadequate by regulators involved in C&L because they do not fulfill to the narrow criteria set to quantify the intrinsic toxicity. There is however a clear difference in the evaluation of a standard aquatic ecotoxicity test and an ecotoxicity test performed using the Bulk approach. In order to class a standard laboratory toxicity study valid, it is of particular importance that – besides information on test substance, test method/conditions and test organism used - suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations are based upon measured levels of the dissolved concentration.

For ecotoxicity tests performed using the bulk approach, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the dose-response relationship should thus be based on the sum of adsorbed and dissolved substance. Results from bulk-approach tests are therefore easier to interpret because nominal concentrations corrected for sorption to glassware can be used to quantify the dose. Because of the use of natural river water a bulk approach test is more environmentally realistic than the standard method and due to that considered to be a higher tier study.

Droge & Goss (2013) have shown that sorption of cationic surfactants to soil and sediment is mainly driven by electrostatic interaction and to a lesser extent by hydrophobic interaction. This means that both the suspended matter and dissolved organic carbon in surface water are the key surface water properties determining the bioavailability of the test item.

The natural surface water was therefore characterized in detail and selected to contain a realistic worst-case suspended matter concentration of 15±3.5 mg/L and ± 3.5 mg/L DOC(≈NPOC). It should be noted that this composition is in perfect alignment with the risk assessment method developed by ECHA, as the concentration of suspended matter in surface water is considered to be 15 mg/L in CHESAR III for risk assessment (see ECHA’s guidance R.16, v3.0, Feb 2016, p. 88).

Sorption to glassware was observed to be <0.08% which means that the nominal test concentrations can be used for the dose-response because the recoveries in the fresh media were in the range of ≥80 % of the nominal values. The difference between the observed recoveries and nominal concentrations is explained by the rapid sorption of AAI-DETA to the river water constituents. Based on the study result, the NOEC for reproduction, mortality, adult length and weight of the test substance for Daphnia magna were determined to be 270 µg/L.

The degree of mitigation of the long-term toxicity to daphnia due to the use of natural river water can be calculated by taking the ratio of the results observed for the bulk approach test and the nominal test results observed for the WAF approach.

The mitigation factor for the chronic effect (NOEC/EC₁₀) to daphnia is 270/133 is 2.03

 

• Guidance to Regulation (EC) No 1272/2008 on classification, Labelling and packaging (CLP) of substances and mixtures


• OECD Guidance document 23 (2019) Aqueous-phase aquatic toxicity testing of difficult test chemicals. Series of testing and assessment No. 23 (second edition).


• Girling AE, Markarian RK, Bennett D (1992) Aquatic toxicity testing of oil products–some recommendations. Chemosphere 24:1469–1472. https://doi.org/10.1016/0045-6535(92)90268-V


• Droge, S.T.J. and Goss, K.W. 2013. Development and Evaluation of a New Sorption Model for Organic Cations in Soil: Contributions from Organic Matter and Clay Minerals. Environmental Science and Technology, 47:14233-14241.


• Di Toro, D 2008 Bioavailability of chemicals in Sediments and soils: toxicological and chemical interactions. SERDP/ESTCP Bioavailability workshop


• van Wijk, D., Gyimesi-van den Bos, M., Garttener-Arends, I., Geurts, M., Kamstra, J., Thomas, P., 2009. Bioavailability and detoxification of cationics, I. Algal toxicity of trimethylammonium salts in the presence of suspended matter and humic acid. Chemosphere 75 (3), 303–309.