Hexanamide, N-(2-ethylhexyl)-3,5,5-trimethyl-

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:

2016-05-15 to 2016-05-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test item Antimussol ZJ

Batch number ESD0017844

CAS number (a.s.) 93820-33-8

EC number 298-613-3

Active substance/ N-(2-Ethylhexyl)-isononan-1-amide / 98.3%
content (certified)

Sum Formula C17H35NO

Density 0.87 g/cm3 (20°C)

Appearance Clear, yellowish, viscous, liquid

(Viscosity 1706 mPa x s (20 °C))

pH 6 – 7 (20 °C, 10 g/L in ethanol/water 1:1)

Water solubility ca. 0.5 mg/L (20 °C)

Stability under Not specified
test conditions

Expiry date July 2017

Recommended storage Cool and dry (2 – 8 °C)

Test Facility Actions

Receipt 2015 03 04 and 2016-01-25 (additional delivery)

Identification parameter Name, batch number, state, consistency, colour and turbidity

Retention sample At least 1 g was retained on 2015-03 04 and 2016-01-25 (additional delivery) and stored at 6 ± 2 °C.

Storage conditions 6 ± 2 °C, protected from light, in the tightly closed original container

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analysed via LC-MS/MS
once within 7 days at the start of an exposure interval (0 hours) and at the end of an exposure interval (48 or 72 hours). Since 100% mortality was observed in the saturated solution on test day 4, the respective measurements in the following sampling intervals were excluded from the results. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000). Details of the analytical method are presented in section 14. The analytical results are presented

Sampling for the analytical monitoring
At the start of the exposure intervals, sampling was carried out after preparation of all test item concentrations.
At the end of the exposure intervals (48 or 72 hours), samples were taken from additional replicates, which were prepared without algae and daphnids and
stored under test conditions.
For the longest exposure interval of 72 hours samples were taken at the start (0 hours) and at the end of the exposure interval (72 hours) once within the
test period.

Range (target)
Recoveries of the test item should be within ± 20% of the initially measured concentrations.

Vehicle:

not specified

Details on test solutions:

Definitive Test
Based on the results of an acute immobilisation test performed at the test facility in 2014 (Noack Study-ID: 130611CL / DAI15534), the definitive reproduction test was carried out with five concentrations of the test item Antimussol ZJ in the range of 1.00 to 100% of a saturated solution, prepared with test medium as specified in Table 4.

Preparation of the saturated solution
A saturated solution with a nominal concentration of 2 mL/L of the
test item was prepared with dilution water (see Table 4) three days prior to the start of each exposure interval (-72 hours). The test item (10 mL/ 5 L) was placed by pipette onto the surface of the dilution water. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer for three days at room temperature. After separation of the phases after 2 hours of standing, the saturated solution was separated by siphon from the bottom and was used as the highest concentration level and as a stock solution for the preparation of further concentration levels. Due to the density of the test item, any undissolved material was expected to float on the surface. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). Since a Tyndall effect was observed, the solution was filtered with a membrane filter (0.2 µm, RC, MACHEREY-NAGEL) prior to use in test. The filter was saturated with the test item dispersion prior to the filtration in order to avoid adsorption during the filtration. The first 25 mL of filtrate were discarded. The filtration was interrupted for ca. 10 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used in the test. During filtration, the filter was always kept covered with the test item dispersion.

Test concentrations
5 concentration levels of the test item in a geometric series with a separation factor of √10, prepared by diluting the saturated solution with dilution water, were tested as follows:
1.00 - 3.16 - 10.0 - 31.6 - 100% of the saturated solution

Control
10 replicates of dilution water without test item

 
Test method
The study was performed with a semi-static test design. A medium renewal 3 times per week (i.e. on Monday, Wednesday and Friday) was sufficient.

Renewal of the test solutions
The test solutions were renewed 3 times per week (after 48 or
72 hours). For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette (see ‘Application’).

Test duration
21 days

Test vessels
Glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity, loosely covered with watch glasses

Test volume
50 mL

Dilution water
Same as the culture medium, therefore no acclimation necessary

Number of daphnids and replicates
10 daphnids in 10 replicates were used for all concentration levels
and the control with one daphnid per replicate, which was held individually.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for
the study. 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.

Application
Per replicate, 50 g test solution were weighed out 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.

Feeding
Daily feeding per test vessel; Pseudokirchneriella subcapitata (0.407 - 0.574 mL) and Desmodesmus subspicatus (0.300 - 0.803 mL) suspension was provided as food corresponding to 0.2 mg C per Daphnia and day.
There was variation according to the density of the algae suspension, but it was the same for all test groups on each feeding day.

Test temperature (target)
20 ± 1 °C

Light intensity (target) M
ax. 20 µEm-2 s-1 (max. 1340 lx)

Photoperiod
16/8 hours light/dark cycle

Aeration
Test vessels were not aerated during the test.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system Daphnia magna STRAUS (Clone 5)

Reason for the selectionof 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 of Clone 5 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 the described conditions.

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. 20 µEm-2  s-1 (max. 1340 lx)

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used. The composition of the culture medium is presented

Culture feeding
The culture 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, with an algae cell density of > 106 cells/mL. The algae are cultured at the test facility.

Origin of the food algae
mmlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Geometric mean measured
test item concentration
[mg/L] fresh old fresh old fresh old
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
2016-04-15 2016-04-18 2016-04-25 2016-04-27 2016-05-02 2016-05-04
987 185 174 Not determined, due to 100% adult mortality
303 Not determined 167 172 182 165
Control 176 183 175 170 177 167

Test temperature:

Geometric mean measured
test item concentration
[mg/L] fresh old fresh old fresh old
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
2016-04-15 2016-04-18 2016-04-25 2016-04-27 2016-05-02 2016-05-04
987 20.3 20.3 Not determined, due to 100% adult mortality
303 Not determined 20.3 20.2 20.8 20.4
Control 20.2 20.3 20.2 20.1 20.4 20.4
The temperature was within the range of 20 ± 1 °C.

pH:

Geometric mean measured
test item concentration
[mg/L] fresh old fresh old fresh old
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
2016-04-15 2016-04-18 2016-04-25 2016-04-27 2016-05-02 2016-05-04
987 7.68 7.69 Not determined, due to 100% adult mortality
303 Not determined 7.09 7.71 7.68 7.44
Control 8.19 7.27 7.57 7.31 7.81 7.18
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:

Dissolved Oxygen Concentration [mg/L]
Geometric mean measured
test item concentration
[mg/L] fresh old fresh old fresh old
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
2016-04-15 2016-04-18 2016-04-25 2016-04-27 2016-05-02 2016-05-04
987 7.89 9.10 Not determined, due to 100% adult mortality
303 Not determined 7.23 8.54 7.30 7.65
Control 8.60 7.44 8.49 7.29 8.77 7.56
The dissolved oxygen concentration was above 3 mg/L.

Nominal and measured concentrations:

Effects on Adult Mortality and Reproduction for all Inserted and for Surviving Parents
(based on the geometric mean measured concentrations of Antimussol ZJ)

Geometric mean
measured
test item
concentrations Adult mortality


[µg/L] [%] Mean SD CV Mean SD CV
987 100 0.01) 0.00 n.a. Not applicable, due to 100% adult mortality
303 100 0.21) 0.63 3162)
100 10 67.71) 18.4 272) 69.6 18.5 272)
28.0 0 98.0 9.92 10 98.0 9.92 10
8.64 0 96.0 5.01 5 96.0 5.01 5
Control 0 93.5 12.3 13 93.5 12.3 13
1) = statistically significant reduction of the reproductive output (Williams multiple sequential t-test procedure, α = 0.05)
2) = biologically significant variation between the test replicates (coefficient of variation > 25%)
n.a. = not applicable, since no juveniles were observed.

Reference substance (positive control):

yes

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

28 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

78.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

100 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Results with reference substance (positive control):

A reference test was conducted as an acute immobilisation test (acc. to AQS P 9/2 and OECD 202) under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility. The results of the most recent test are presented

Reference item Potassium dichromate p.a. (SIGMA)

Purity 99.0%

Batch No. MKBV0900V

Storage stability 2016-12-16

Test concentrations 1.00 - 2.00 - 4.00 mg/L

Ranges of validity EC50 (24 hours): 0.6 - 2.4 mg/L, according to AQS P 9/2 (clone 5)
EC50 (24 hours): 0.6 - 2.1 mg/L, according to OECD 202 (clone A)

Test duration 24 hours

Test vessels Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Volume of test medium 20 mL

Dilution water ISO test water, according to OECD 202, Annex 3

Loading of test organisms 20 animals, divided into 4 replicates, each with 5 animals

Test duration 2016-04-05 to 2016-04-06

Reported statistics and error estimates:

 

Statistical evaluation           

     The NOEC and LOEC for the reproduction and the adult mortality were determined with the software ToxRat Professional as specified below. The EC-values for the reduction of the reproductive output and LC-values for the adult mortality and the respective 95% confidence limits were calculated with the software ToxRat Professional. 
Significant deviations 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), trend analysis (Trend analysis by contrasts (monotonicity of concentration-response) and a multiple test (William’s test).
Prior to running the multiple test, a normality test, a variance homogeneity test and a trend analysis were performed. P‑values of the normality and the variance homogeneity test were 0.01 and 0.05 for the trend analysis. Thea-value (acceptable probability of incorrectly concluding that there is a difference) wasa= 0.01 for the normality test and the variance homogeneity test anda= 0.05 for the trend analysis.

                                              For the effects on reproduction, the juveniles per inserted parent at the start of the test excluding accidental and inadvertent mortality (reproductive output) were calculated. Since no accidental and inadvertent mortality was observed, no parental daphnids were excluded from the evaluation of the reproductive output. The effects on reproduction per survived parental animal at the end of the exposure were also calculated.

 

            For the determination of significant deviations of the adult mortality,

                           Step-down Cochran-Armitage test procedure was used. Details of the statistical analyses are presented

  The coefficients of variation around the mean number of living offspring produced per inserted parent and per survived parent in the control and the test groups were evaluated.
Alleffect levels (NOEC, LOEC,EC_{10 / 50 / 100}andLC_{20 / 50 / 100}) given are based on the geometric mean measured test item concentrations, since themeasured test item concentrations at the end of the exposure intervals were not within± 20% of the initially measured concentrations.

 

Determination of the     

EC-/LC-values  

       

   The EC_{10 / 50}-values for the reproductive output and their 95%

                       confidence limits were calculated by point estimates from the 3‑parametric normal CDF with the software ToxRat Professional.
The LC_{20 / 50}-values for the adult mortality and their 95% confidence limits were calculated by probit analysis with the software ToxRat Professional. The LC₁₀₀was empirically derived from the observed data.
The EC₅₀-value for the reference item was calculated by sigmoidal dose-response regression.The respective 95% confidence limits for the EC₅₀-value of the reference item were calculated from the standard error and the t-distribution. These calculations were carried out from the best-fit values with the software GraphPad Prism5.

 

Calculation of the      

intrinsic rate            

           

   The intrinsic rate of natural increase (IR) was calculated for the

                     survived parental daphnids with the followingEuler-Lotka-equation with the ToxRatProfessional.

                                              S(e^-A*IR) * S_x* J_x= 1

                                                              A                      = Age

                                                              IR                     = Intrinsic rate of natural increase

                                                              S_x                    = Survival rate on day x

                                                              J_x                    = 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:

- Excel,Microsoft Corporation
- GraphPad Prism, GraphPad Software, Inc.
- SigmaPlot, Systat Software, Inc.
-ToxRat Professional, Version 3.2.1,ToxRat Solutions GmbH

The number of juveniles in all replicates of the control and the test groups was counted and recorded every day. Results concerning the number of living juveniles of all concentration levels and the control for every test day are presented inTable13toTable18.

The average number of living juveniles at the end of the test after 21 days per survived parental daphnid was 94 in the control group and thus thevalidity criterion for the reproduction of a mean of ≥ 60 living offspring per survived parent animalin the control was met.

No parental daphnids were excluded due to accidental or inadvertent mortality from the evaluation of the reproductive output. Since the Cochran-Armitage test procedure revealed a significant increasing trend (p≤0.05) in adult mortality, the evaluation was done based on the cumulative offspring per inserted parent. A statistically significant reduction of the reproductive output in comparison to the control was determined at the concentration levels 100 to 987 µg/L (Williams multiple sequential t-test procedure,α = 0.05). The determination of the EC₁₀- and the EC₅₀-value and their 95% confidence limits was carried out by point estimates from the 3-parametric normal CDF.

The EC₁₀for the test itemAntimussolZJwas78.7 µg/L (95% confidence limits: 38.0 - 162 µg/L).The EC₅₀was 119µg/L (95% confidence limits: 28.6 - 467 µg/L).The LOEC was 100 µg/L. The NOEC was 28.0 µg/L.

 

 the parameter living offspring per survived parental animal at the end of exposure was also evaluated and the results were comparable to the cumulative offspring per inserted parent animal. Nevertheless, the evaluation based on the cumulative offspring per inserted parent is the ecologically most relevant response variable.

 

 

 Adult Mortality

 

The test item induced anadult mortalityof 100% in the concentration levels 303 and 987 µg/L and 10% in the concentration level 100 µg/L during the test period of 21 days.In the control as well as in the concentration levels 8.64 and 28.0 µg/L, all parental daphnids survived until the end of the test after 21 days. Accidental and inadvertent mortality as defined in the OECD test guideline were not observed.

The adult mortality was statistically significant in the concentration levels 303 and 987 µg/L (Step‑down Cochran-Armitage Test Procedure,α = 0.05). The Cochran-Armitage test procedure revealed a significant increasing trend (p≤0.05) in adult mortality. Therefore, the LC₂₀- and LC₅₀-values were calculated by probit analysis.

The LC₂₀for the test itemAntimussolZJwas 104µg/L (95% confidence limits: 68.2 - 138 µg/L).The EC₅₀was 152µg/L (95% confidence limits: 113 - 206 µg/L).The LOEC was 303 µg/L. The NOEC was 100 µg/L. For details, seeTable1,Table2,Table5andFigure4.

 

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

                (n = 10)

Geometric mean measured test item concentrations [µg/L]		Adult Mortality [%]
		7 days		14 days		21 days
987		100		100		100^1)
303		80		90		100^1)
100		0		0		10
28.0		0		0		0
8.64		0		0		0
Control		0		0		0

¹⁾= statistically significant (Step‑down Cochran-Armitage Test Procedure,α = 0.05)

 

 

   Mortalityof the Adult Daphnids after 21 days

                     (based on the geometric mean measured concentrations ofAntimussol ZJ[µg/L])

 

      Intrinsic Rates of Natural Increase (IR)

 

Theintrinsic rates of natural increase (IR)of the survived parental daphnids accounting for generation time and number of offspring were used for calculation of population growth and maintenance. For details, seeTable3andTable6.

 

 Intrinsic Rates of Natural Increase

Geometric mean measured test item concentrations [µg/L]		Intrinsic rate of natural increase in replicate no.											Mean IR			CV
		1		2		3		4		5		6		7		8		9		10		MV±SD			[%]
987		--		--		--		--		--		--		--		--		--		--		--				--
303		--		--		--		--		--		--		--		--		--		--		--				--
100		0.336		0.253		0.276		0.400		--		0.317		0.334		0.343		0.336		0.291		0.321±		0.0431		13.4
28.0		0.400		0.347		0.380		0.405		0.408		0.372		0.363		0.363		0.371		0.345		0.375±		0.0227		6.0
8.64		0.338		0.401		0.369		0.373		0.351		0.384		0.337		0.353		0.377		0.379		0.366±		0.0208		5.7
Control		0.423		0.362		0.332		0.343		0.345		0.361		0.368		0.329		0.305		0.350		0.352±		0.0312		8.9

--   = not applicable, due to the mortality of the parental daphnid(s)

 

 

     Number of Broods and first Appearance of living Juveniles

 

At the end of the test after 21 days, the number of broods with living juveniles produced by the survived parental daphnids was 4 in the control and in the concentration levels 8.64 to 100 µg/L. However, one parental daphnid at the concentration level 28.0 µg/L produced 5 broods with living juveniles. In the concentration level 100 µg/L, two parental daphnids produced only 3 broods with living juveniles, since broods occurred delayed. In the two highest concentration levels 303 and 987 µg/L, all parental dapnids died during the exposure period.
Thefirst appearance ofliving juveniles was observed on days 8 and 13 at all survived parental daphnids of the control and the concentration levels 8.64 to 100 µg/L.For details, seeTable3andTable7.

 

  First Appearance of Living Juveniles and Mean Number of Broods in the Individual Groups

Geometric mean measured test item concentrations [µg/L]		Day of first appearance of living juveniles at the survived parental daphnids in replicate no.											First		Mean
												appearance		number
		1		2		3		4		5		6		7		8		9		10		mean day		of broods*
987		--		--		--		--		--		--		--		--		--		--		--		--
303		--		--		--		--		--		--		--		--		--		--		--		--
100		9		13		10		9		9		9		9		9		9		9		9.5		3.8
28.0		8		8		8		8		8		8		9		9		9		9		8.4		4.1
8.64		9		8		9		9		9		8		9		9		8		8		8.6		4.0
Control		8		9		9		9		9		8		9		9		11		9		9.0		4.0

--   = not applicable, due to the mortality of the parental daphnid(s)

*    = only broods of the survived parental daphnids with living juveniles taken into account

 

 

     Stillborn Juveniles and Aborted Eggs

 

Nostillborn juvenilesoraborted eggswere observed in the control and in the concentration level 28.0 µg/L during the exposure period of 21 days (seeTable8andTable9). Related to the total number of produced juveniles (dead + alive) per survived daphnid, the percentage of dead juveniles was≤4% in the concentration levels 8.64 and 100 µg/L (Table3).

 

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

Geometric mean measured test item concentrations [µg/L]		Number of			Total no.   å		Number of survived adults N
		stillborn juveniles		aborted eggs
987		--		--		--		0
303		--		--		--		0
100		22		7		29		9
28.0		0		0		0		10
8.64		1		0		1		10
Control		0		0		0		10

N   = number of the survived parental daphnids

--   = not applicable, due to the mortality of the parental daphnid(s)

 

 

      Relative Number of Dead (Stillborn + Aborted Eggs)to Total Number of Juvenilesper Survived Parental Daphnids

Geometric mean measured test item concentrations [µg/L]		Number of juveniles				Percentage of dead juveniles^#) [%]
		Dead		Alive			Total
987		--		--		--		--
303		--		--		--		--
100		29		626		655		4
28.0		0		980		980		0
8.64		1		960		961		< 1
Control		0		935		935		0

Dead   = aborted eggs + stillborn juveniles

Total  = dead + alive juveniles

^#)          = related to the total number of juveniles

--         = not applicable, due to the mortality of the parental daphnid(s)

 

 

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

 

The mean values of the body length (excluding the anal spine) of the survived parental daphnids in the tested concentration levels 8.64 to 100 µg/L were in the range of 4.64 to 4.78 mm per daphnid and 4.78 mm per daphnid in the control group.

The mean dry weight of the survived parental daphnids was in the range of 0.678 to 0.940 mg per daphnid in the concentration levels 8.64 to 100 µg/L and 0.900 mg per daphnid in the control

 

   Total Body Length and Dry Weight of the Survived Parental Daphnids

Geometric mean measured test item concentrations [µg/L]	Total length of the survived parent animals [mm]										MV	N	Dry weight [mg]
	Replicate no.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
987	--	--	--	--	--	--	--	--	--	--	--	0	--
303	--	--	--	--	--	--	--	--	--	--	--	0	--
100	4.50	4.50	4.50	4.75	--	4.75	4.75	4.75	4.75	4.50	4.64	9	6.1	0.678
28.0	4.75	4.75	4.50	4.50	4.75	4.75	4.75	4.75	4.75	4.75	4.70	10	9.0	0.900
8.64	4.75	4.75	4.75	4.75	4.75	4.75	4.75	4.75	4.75	5.00	4.78	10	9.4	0.940
Control	4.75	4.75	4.75	4.75	5.00	4.75	4.75	4.75	4.75	4.75	4.78	10	9.0	0.900

N  = number of the survived parental daphnids

--   = not applicable, due to the mortality of the parental daphnid(s)

 

 

     Presence of Males

 

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

 

 

  Occurrence of Ephippia (Winter Eggs)

 

No ephippiawere observedin the control or in the test groups during the test.

 

 

Validity criteria fulfilled:

yes

Conclusions:

The effects on reproduction were evaluated based on the reproduction per inserted parent animal, since this parameter is the ecologically most relevant response variable and is required by the OECD test guideline 211 (2012), when a significant trend in mortality is detected.
A summary of the assessed effect levels based on the geometric mean measured concentrations of the test item Antimussol ZJ is given in the table below.
The overall effect threshold for effects of the test item under the test conditions was 28.0 µg/L (NOEC) and 100 µg/L (LOEC) based on the geometric mean measured concentrations of the test item Antimussol ZJ.

Table 1: Endpoints for Reproduction and Mortality
(based on the geometric mean measured test item concentrations)
Effect values Antimussol ZJ
Geometric mean measured
test item concentrations
[µg/L]
EC10 Reproduction
(with 95% confidence limits) 78.7 (Cl: 38.0 - 162)
EC50 Reproduction
(with 95% confidence limits 119 (Cl: 28.6 - 467)
LOEC Reproduction 100
NOEC Reproduction 28.0
LC20 Adult mortality after 21 days
(with 95% confidence limits) 104 (Cl: 68.2 - 138)
LC50 Adult mortality after 21 days
(with 95% confidence limits) 152 (Cl: 113 - 206)
LC100 Adult mortality after 21 days 303
LOEC Adult mortality after 21 days 303
NOEC Adult mortality after 21 days 100

Executive summary:

A Daphnia magna reproduction test (semi-static, 21 d) of the test itemAntimussol ZJ (batch number:ESD0017844) was conducted according to OECD 211 (2012) from2016-04-12 to 2016‑05‑06, with the definitive exposure phase from 2016-04-15 to 2016-05-06, at the test facility.

Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids, held individually, were used per concentration level and control. At test start the daphnids were 2 to 24 hours old. The study was carried out undersemi-static conditions with a medium renewal three times per week (i.e. on Monday, Wednesday and Friday). 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.

The test item Antimussol ZJ is a clear, yellowish, viscous liquid with a water solubility of approximately 0.5 mg/L. Nominal concentrations of the test itemAntimussolZJwere selected based on the results of an acute immobilisation as follows:1.00–3.16–10.0–31.6–100% of the saturated solution.

A saturated solution with a nominal concentration of 2 mL test item/L was prepared with dilution water (Table4) as specified in section 4.2 and was used as the highest concentration level and as a stock solution for the preparation of a further four concentration levels in the range of 1.00 to 31.6% of the saturated solution prepared with dilution water out of the saturated solution in a geometric series with a separation factor of√10.

The test item concentrations ofAntimussol ZJwere analytically verified via LC-MS/MSat the start of the exposure intervals on days 0, 12, 17 (0 hours) and at the end of the exposure intervals on days 3, 14, 19 (48 or 72 hours, respectively) in all concentration levels with surviving parental daphnids and the control. Details of the analytical method are presented.

The measured concentrations of the test item at the start of the respective exposure intervals (0 hours) reflect the separation factor of√10between the test concentrations. At the end of the respective exposure intervals (48 and 72 hours), the measured concentrations were in the range of 68 to 109% of the initially measured concentrations.The analytical results are presented

Since the measured test item concentrations at the end of the exposure intervals were not within± 20% of the initially measured concentrations, the geometric mean measured concentrations were calculated and used for estimation of the effect-values. The geometric mean measured concentrations are: 8.64 - 28.0 - 100 - 303 - 987 µg/L.

The environmental conditionswere within the acceptable limits.The validity criteria of the test guideline were met.

The effects on reproduction were evaluated based on the reproduction per inserted parent animal. A summary of all endpoints based on the geometric mean measured concentrations of the test itemAntimussol ZJ is given.

 

 

 Endpoints for Reproduction and Mortality

(based on the geometric mean measuredtest item concentrations)

Effect values	AntimussolZJ Geometric mean measured test item concentrations [µg/L]
EC10 Reproduction (with 95%confidence limits)	78.7	(Cl: 38.0 - 162)
EC50 Reproduction (with 95%confidence limits	119	(Cl: 28.6 - 467)
LOECReproduction	100
NOECReproduction	28.0
LC20 Adult mortality after 21 days (with 95%confidence limits)	104	(Cl: 68.2 - 138)
LC50 Adult mortality after 21 days (with 95%confidence limits)	152	(Cl: 113 - 206)
LC100 Adult mortality after 21 days	303
LOECAdult mortality after 21 days	303
NOECAdult mortality after 21 days	100

 

 

Reproductive output:

 

No parental daphnids were excluded due to accidental or inadvertent mortality from the evaluation of the reproductive output. Since the Cochran-Armitage test procedure revealed a significant increasing trend (p≤0.05) in adult mortality, the evaluation was done based on the cumulative offspring per inserted parent. A statistically significant reduction of the reproductive output in comparison to the reproductive output in the control was determined at the concentration levels 100 to 987 µg/L (Williams multiple sequential t-test procedure,α = 0.05). The determination of the EC₁₀- and the EC₅₀-value and their 95% confidence limits was carried out by point estimates from the 3-parametric normal CDF.

The EC₁₀for the test itemAntimussolZJwas78.7 µg/L (95% confidence limits: 38.0 - 162 µg/L).The EC₅₀was 119µg/L (95% confidence limits: 28.6 - 467 µg/L).The LOEC was 100 µg/L. The NOEC was 28.0 µg/L.

 

Adult mortality:

 

Accidental and inadvertent mortality was not observed. The test item induced statistically significant adult mortality of 100% in the concentration levels 303 and 987 µg/L (Step‑down Cochran-Armitage Test Procedure,α = 0.05). In the concentration level 100 µg/L, a mortality rate of 10% of the inserted adults was observed, which is not statistically significant. In all other concentration levels and in the control all parental daphnids survived until the end of the test (21 days). The Cochran-Armitage test procedure revealed a significant increasing trend (p≤0.05) in adult mortality. Therefore, the LC₂₀- and LC₅₀-values were calculated by probit analysis.

The LC₂₀for the test itemAntimussolZJwas 104µg/L (95% confidence limits: 68.2 - 138 µg/L).The EC₅₀was 152µg/L (95% confidence limits: 113 - 206 µg/L).The LOEC was 303 µg/L. The NOEC was 100 µg/L. For details, seeTable1,Table2,Table5andFigure4.

 

   Effects on Adult Mortality and Reproduction for all Inserted and for Surviving Parents

(based on the geometric mean measuredconcentrations ofAntimussol ZJ)

Geometric mean measured test item concentrations	Adult mortality	Mean number of offspring per inserted parental daphnid			Mean number of offspring per survived parental daphnid
[µg/L]	[%]	Mean	SD	CV	Mean	SD	CV
987	100	0.0^1)	0.00	n.a.	Not applicable, due to 100% adult mortality
303	100	0.2^1)	0.63	316^2)
100	10	67.7^1)	18.4	27^2)	69.6	18.5	27^2)
28.0	0	98.0	9.92	10	98.0	9.92	10
8.64	0	96.0	5.01	5	96.0	5.01	5
Control	0	93.5	12.3	13	93.5	12.3	13

¹⁾      = statistically significant reduction of the reproductive output (Williams multiple sequential t-test procedure, α = 0.05)

²⁾      = biologically significant variation between the test replicates (coefficient of variation > 25%)

n.a.  = not applicable, since no juveniles were observed.

 

 

Further effects:A summary of other biological effects is presented inTable3.

 

    Other Observed Biological Effects based on Surviving Parental Daphnids

Geometric mean measured test item concentrations	Mean intrinsic rate	Age at first brood	Mean number of broods	Dead juveniles related to the total number of juveniles	Mean body length	Mean dry weight
[µg/L]	[1/d]	[Mean Day]	[N]	[%]	[mm]	[mg]
987	Not applicable, due to 100% adult mortality
303
100	0.321	9.5	3.8	4	4.64	0.678
28.0	0.375	8.4	4.1	0	4.70	0.900
8.64	0.366	8.6	4.0	< 1	4.78	0.940
Control	0.352	9.0	4.0	0	4.78	0.900

 

Description of key information

The effects on reproduction were evaluated based on the reproduction per inserted parent animal, since this parameter is the ecologically most relevant response variable and is required by the OECD test guideline 211 (2012), when a significant trend in mortality is detected.

A summary of the assessed effect levels based on the geometric mean measured concentrations of the test item Antimussol ZJ is given in the table below.

The overall effect threshold for effects of the test item under the test conditions was 28.0 µg/L (NOEC) and 100 µg/L (LOEC) based on the geometric mean measured concentrations of the test item Antimussol ZJ.

Table 1:       Endpoints for Reproduction and Mortality

(based on the geometric mean measured test item concentrations)

Effect values       Antimussol ZJ

Geometric mean measured

test item concentrations

[µg/L]

EC10 Reproduction

(with 95% confidence limits)         78.7       (Cl: 38.0 - 162)

EC50 Reproduction

(with 95% confidence limits       119       (Cl: 28.6 - 467)

LOEC Reproduction       100       

NOEC Reproduction         28.0       

LC20 Adult mortality after 21 days

(with 95% confidence limits)       104       (Cl: 68.2 - 138)

LC50 Adult mortality after 21 days

(with 95% confidence limits)       152       (Cl: 113 - 206)

LC100 Adult mortality after 21 days       303       

LOEC Adult mortality after 21 days       303       

NOEC Adult mortality after 21 days       100       

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

78.7 µg/L