Dodecanoic acid, ester with 1,2,3-propanetriol

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 October - 26 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, 31 May 2016

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Test item concentrations were measured at fresh and aged test solutions at each media renewal. The content of the test item was determined by using the mono dodecanoic acid ester for the analytical measurements because of its liquid chromatographic properties.
- Sampling method: Samples of fresh test solutions were taken from the test solution preparations after distributing it to the 10 replicates. For aged test solutions samples were taken from a representative test vessel per concentration. Sampling was performed at each media renewal. A sample volume of 5 mL of each sample were transferred in 15 mL polypropylene (PP) centrifugation tubes prepared with 5 mL methanol. The tubes were shaken to homogenize the solutions. The sample-methanol mixtures of the samples were kept frozen at -20 °C until analysis. If necessary the samples were diluted with methanol / copper-free water (50:50 v:v) and measured by LC-MS/MS.
- Sample storage conditions before analysis: Samples were stored until analysis under circumstances to maintain the test item stable to the best of knowledge until analysis, but were measured at least seven days after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock and all test item solutions were prepared in Cu-reduced dilution water which was sterilized by filtration through a 0.22 µm PES filter before use. Before test start and before each media renewal a stock solution was prepared by weighing in an adequate amount of test item to reach a nominal loading of 10 mg/L and transferring it to sterilized Cu-free dilution water. Directly after addition of the test item the stock solution was sonicated for one hour in a sonication bath (Bandelin, RK510; Frequence 35 kHz) at about 30°C. Afterwards the stock solution was stirred for further 2 hours at room temperature. The test concentrations were prepared by dilution of the stock solution with sterilized Cu-free dilution water. Individual dilutions from the stock solution was prepared. The test solutions were freshly prepared before test start and at each medium renewal daily.
- Other: In four pre-tests, different approaches for preparation of test solutions were performed. Recovery rates of the test item from different approaches were compared to identify the most suitable application procedure. The solubility of the test item was analyzed to reach approximately nominal concentrations in aqueous solutions for the main test. For this, the test solutions were generated by stirring, ultrasonification, different temperatures, different time ranges or organic solvents and a combination of these options, respectively. All test solutions of the pretests were diluted to generate the analytical samples in MeOH / copper-free water (50:50). The recoveries are based on the aqueous part of the analytical samples. Based on the results of the performed pre-tests, this approach for preparation of test item concentrations was chosen.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene
- Age of parental stock: 4 – 24 hours
- Feeding during test
- Food type: Mixture of suspensions of unicellular alga Desmodesmus subspicatus
- Amount: The content of food in the test suspensions, measured at 585 nm, was kept constant at 0.2 mg C/(Daphnia * day).
- Other: Specimens used in the test are bred in the laboratory of the Fraunhofer IME.

ACCLIMATION
- Acclimation period: one week
- Acclimation conditions (same as test or not): Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving. Batches of 30 to 50 animals were held at room temperature in ca. 1.8 L dilution water for one week. The water was changed once per week. Newborn D. magna were separated by sieving, the first generation was discarded.
- Type and amount of food: During this one week the daphnids were fed with an algal suspension (Desmodesmus subspicatus) and ArtemioFluid (JBL). Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a few mL of medium. 30 mL of this suspension was given to 1 L Daphnia medium.
- Feeding frequency: daily
- Health during acclimation: Individuals applied in the test were transferred with a bore Pasteur pipette a few hours after sieving to ensure applying only healthy specimens.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

70 – 120 mg/L as CaCO3

Test temperature:

20.0 – 20.5 °C

pH:

7.00 – 8.54

Dissolved oxygen:

3.14 - 9.03 mg/L

Nominal and measured concentrations:

nominal: 0.01, 0.032, 0.1, 0.316 and 1.0 mg/L
time weighted mean (TWM): 0.003, 0.008, 0.026, 0.087 and 0.315 mg/L (corresponding with 25.7, 25.5, 26.4, 27.6 and 31.5 % of nominal concentrations)

Details on test conditions:

TEST SYSTEM
- Test vessel: Numbered glass Erlenmeyer flasks 100 mL nominal volume
- Type: The beakers were covered with air-permeable silicone-sponge caps to prevent from evaporation as much as possible, but also permit gaseous exchange between the medium and the atmosphere and access of light.
- Material, size, headspace, fill volume: 100 mL test solution
- Aeration: no
- Renewal rate of test solution: Daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Other: To avoid copper contamination, plastic water pipes were used for the testing facilities. The vessels and caps were sterilized prior to use (autoclaving). test was performed under sterile conditions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Cu-reduced dilution water
- Culture medium different from test medium: no
- Intervals of water quality measurement: Oxygen concentration, pH value and temperature were checked at each media renewal in new and aged test solutions of the control and each test concentration. Measurements of these water parameters was performed in new test media after distributing the test solutions to the replicates in an additional test vessel and in aged media in pooled samples for the control and for each treatment level. Hardness was checked once a week at water renewal in new and aged media of the control and the highest test concentration.

OTHER TEST CONDITIONS
- Photoperiod: light/dark cycle of 16/8 hours
- Light intensity:720 – 966 lx (corresponding to 9.6 – 12.9 µE/(m² *s)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The numbers of immobile daphnids were visually determined daily and the immobile daphnids were removed. Any abnormalities in appearance and behaviour were recorded. The newborn daphnids per beaker were counted and removed daily. Any abnormalities in condition (including male sex) or presence of winter eggs were checked and recorded. At study termination, length of the adults without significant mortality was measured by digital photography and image analysis and compared with the equally measured control animals.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study
- Test concentrations: 0.01, 0.1 and 1.0 mg/L
- Results used to determine the conditions for the definitive study: In the range-finding test a dose dependent effect of the test item on the reproduction of survived parent daphnids was observed. After 21 days, a significant reduction of the reproduction of 20 % was observed in the second test concentration of 0.1 mg/L and a significant reduction of 75 % was observed in the highest test concentration of 1.0 mg/L compared to the control. No significant effect on reproduction was observed in the first test concentration of 0.01 mg/L. In the highest test concentration of 1.0 mg/L two daphnids died during the 21 day exposure period, resulting in an immobility of 33.3.%. In the second concentration of 0.1 mg/L one daphnid died, resulting in 16.7 % immobility. In the lowest concentration of 0.01 mg/L and in the control no daphnid died during the test. The LC50 for immobility was calculated to be 2.56 mg/L.
The test was conducted under semi-static and sterile conditions for 21 days. Renewal of test solutions was conducted daily.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.027 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: single replicates

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.054 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: mean number of replicates

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.087 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: single replicates/ mean number of replicates

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.525 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: single replicates

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.404 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: mean number of replicates

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.003 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: length

Remarks on result:

other: single replicates/ mean number of replicates

Details on results:

- Behavioural abnormalities: Daphnids showed no signs of discoloration or abnormalities in their behavior. No other clinical signs were observed in any replicate at any concentration tested.Neither any physical nor pathological symptoms were obtained.
- Observations on body length: A significant difference in the adult body length of individuals was shown between the control and the second and higher test concentrations (see table).
- Mortality of control: no mortality

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50(24h) = 1.435 mg/L (95% CL :1.23 – 1.75)
- Other: The results of the latest reference study (November 2017) are in agreement with historical 24 h EC50-values obtained in this institute.

Reported statistics and error estimates:

Probit anaylsis

During the 21 day exposure period to the test item Dodecanoic acid, ester with 1,2,3-propanetriol significant effects on the reproductive performance and on the length of the test organisms Daphnia magna were observed.

Age at the first brood was between 9.8 and 10.9 days for control and the treatments. The difference was not statistically significant.

The five test concentrations showed time weighted mean measured test item concentrations of freshly prepared test solutions between 56.4 – 63.2 % of nominal concentrations. During the time interval until renewal of the test solution, test item concentrations decreased to 8.4 – 14.9 % of nominal. Since the deviation of the mean measured test concentration was greater than ± 20% of nominal concentrations, the evaluation of the effects was performed on basis of the time weighted mean test concentrations (TWA).

Table 1:         Survival data and percent reduction of survival compared to the control after 21 days.

TWM Conc. [mg/L]	Parental survival	Reduction of survival [%] (immobility)
Control	10	-
0.003	9 (-)	10 (-)
0.008	10 (-)	0 (-)
0.026	10 (-)	0 (-)
0.087	9 (-)	10 (-)
0.315	9 (-)	10 (-)

(+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments

 

Table 2:         Reproduction data.

TWM conc. [mg/L]	Cumulative offspring per survived parent Mean±SD [Ind.]	Cumulative offspring per introduced parent Mean± SD [Ind.]	Age of first brood Mean± SD [days]	Development rate Mean± SD [Ind.]	Intrinsic rate Mean± SD [Ind./day]
Control	77.1± 12.35	77.1±12.35	10.9± 0.97	0.092± 0.008	0.284± 0.014
0.003	68.9± 12.64 (-)	67.6± 12.60 (-)	10.5± 0.47 (-)	0.095± 0.004 (-)	0.289± 0.012 (-)
0.008	82.7± 14.35 (-)	82.7± 14.35 (-)	9.9± 0.84 (-)	0.102± 0.009 (-)	0.312± 0.027 (-)
0.026	70.0± 13.06 (-)	70.0± 13.06 (-)	9.8± 0.82 (-)	0.103± 0.008 (-)	0.297± 0.007 (-)
0.087	67.4± 10.35 (-)	67.0± 9.85   (-)	9.9± 0.70 (-)	0.101± 0.008 (-)	0.292± 0.015 (-)
0.315	42.6± 14.59 (+)	42.5± 13.75 (+)	10.2± 1.25 (-)	0.099± 0.010 (-)	0.272± 0.014 (-)

(+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments; SD: standard deviation

 

Table 3:        Percent reduction of reproduction compared to controls after 21 days.

TWM conc. [mg/L]	Reduction of reproduction per survived parent [%]	Reduction of reproduction per introduced parent [%]	Inhibition of development rate [%]	Inhibition of intrinsic rate [%]
Control	-	-	-	-
0.003	10.6(-)	12.3 (-)	-3.3 (-)	-1.8 (-)
0.008	-7.3 (-)	-7.3 (-)	-10.1 (-)	-10.0 (-)
0.026	9.2 (-)	9.2 (-)	-11.1 (-)	-4.7 (-)
0.087	12.5 (-)	13.1 (-)	-9.8 (-)	-3.1 (-)
0.315	44.8 (+)	44.9 (+)	-7.3 (-)	4.1 (-)

(+) statistically significant difference between controls / (-) no significant difference between controls and treatments

Table 4:         Growth data and percent reduction of length compared to controls after 21 days.

TWM Conc. [mg/L]	Length on day 21 Mean±SD [mm]	Decrease in length [%]
Control	4.41±0.15	-
0.003	4.32± 0.15(-)	2.1 (-)
0.008	4.26± 0.13(+)	3.4 (+)
0.026	4.17±0.24 (+)	5.5 (+)
0.087	4.21± 0.19(+)	4.6 (+)
0.315	4.11± 0.23(+)	6.9 (+)

(+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments; SD: standard deviation

 

Table 5:         Measured concentrations of test item.

Test item nominal concentration [mg/L]	Mean measured initial concentration [mg/L]	% of nominal
Control	< LOQ*	-
0.01	0.006	59.5
0.03	0.019	58.1
0.10	0.056	56.4
0.32	0.187	59.2
1.00	0.632	63.2
	Mean measured aged concentration [mg/L]	% of nominal
Control	< LOQ*	-
0.01	0.001	8.4
0.03	0.003	9.5
0.10	0.010	10.2
0.32	0.033	10.4
1.00	0.149	14.9
	Time weighted mean concentration [mg/L]	% of nominal
Control	< LOQ*	-
0.01	0.0026	25.7
0.03	0.0076	25.5
0.10	0.0264	26.4
0.32	0.0871	27.6
1.00	0.3145	31.5

* LOQ = 0.001 mg/L

Table 6: Validity criteria for OECD 211.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals (female Daphnia) does not exceed 20% at the end of the test.	0% mortality was recorded in the control after 21d.	yes
The mean number of living offspring produced per parent animal surviving at the end of the test is ≥ 60.	The mean number of living offspring produced per parent animal surviving at the end of the test is 77.1± 12.35.	yes

Note: The same validity criterion (20%) can be used for accidental and inadvertent parental mortality for the controls as well as for each of the test concentrations.

 

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

EC10 (21 d) = 0.027 mg/L (TWA) for Daphnia magna (OECD 211)

NOEC (21d) = 0.087 mg/L (TWA) for Daphnia magna (OECD 211)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.027 mg/L

Additional information

One study investigating the chronic toxicity of Dodecanoic acid, ester with 1,2,3-propanetriol (CAS 37318-95-9) to Daphnia magna is available. This semi-static test was conducted according to OECD 211 under GLP conditions with daily renewal of test solution. Daphnia magna was exposed for 21 d to five test concentrations of 0.01, 0.032, 0.1, 0.316 and 1.0 mg/L (nominal). Analytical measurements were conducted for all test concentrations at fresh and aged test solutions at each media renewal. In pre-tests, different approaches for preparation of test solutions were performed to identify the most suitable application procedure for comparable recovery rates. The five test concentrations showed time weighted mean measured test item concentrations of freshly prepared test solutions between 56.4 – 63.2 % of nominal concentrations. During the time interval until renewal of the test solution, test item concentrations decreased to 8.4 – 14.9 % of nominal. Thus, the effect concentrations were based on time weighted average (TWA) concentrations resulting in 0.003, 0.008, 0.026, 0.087 and 0.315 mg/L. For the assessment of effects and for calculation of effect concentrations the number of offspring per single replicate was used. There were significant effects on the reproduction of Daphnia magna over the range of the tested concentrations. The difference of the number of offspring between the control and the highest test concentration of 0.315 mg test item/L was statistically significant. In addition, reduction of reproduction was > 10% at 0.087 mg/L test concentration. Thus, based on the number of cumulative offspring per survived parent the EC10 was calculated to be 0.027 mg/L (meas. TWA) and a NOEC of 0.087 mg/L (meas. TWA) was determined.