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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 October 2018 to 24 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
Standard GUIDELINE OECD 211 (2012)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch number 1700001162
- Expiration date of the lot/batch: 12 June 2020
- Purity test date: 20 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in Darkness at a temperature of 20°C
- Stability under test conditions:The stability (non-GLP) was checked for reference and test items stock solutions after respectively 20 and 16 days at -20°C +/- 5°C. Recoveries were higher than 90%.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:Since the test item is poorly soluble under the test conditions and according to the OECD 23, the choice of a solvent was necessary to solubilize the test material.A stock solution in dimethyl sulfoxide was prepared and test solutions were prepared by dilution of this solution.
- Final dilution of a dissolved solid, stock liquid or gel:A stock solution in dimethyl
sulfoxide (DMSO) was prepared at 20 g test item.L-1.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
The Test material was applied as liquid after solubilization in dimethyl sulfoxide (DMSO)

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Test limit was performed at 2 mg of test item.L-1
- Sampling method: Test item solution and control were sampled and immediately analysed
- Sample storage conditions before analysis: No storage before analysis
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:Dilution of the stock solution prepared by dissolving 200 mg of test material in 10 mL of dimethyl sulfoxide(DMSO), concentration of stock solution = 20 g/L
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethyl sulfoxide
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):According to the OECD monograph 23, the solvent concentration was not exceeded 0.1 mL.L-1 in the test and control solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna Strauss
- Strain/clone:Strauss clone 5
- Age at study initiation: between 6 and 24 hours old.
- Method of breeding: The test organism was produced from parthenogenically reproducing brood female population. Daphnia are issued from the breeding lab. The juvenile stemming at least from the second litter were used to start the test.
- Source: Daphnia are issued from the breeding lab.
- Feeding during test: Yes
- Food type: Pseudokirchneriella subcapitata and Chlorella vulgaris suspensions .
- Amount: 0.1 mg of Carbon / daphnia / day
- Frequency: Daily feeding
Culture conditions:
Medium: OECD Elendt M4
Temperature: 20 °C ± 2
Light/dark cycle: 16 h/8 h
pH: Between 6 and 9
Hardness (CaCO3): Between 140 and 250 mg.L-1

Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
Hardness (CaCO3): Between 140 and 250 mg.L-1
Test temperature:
Temperature (°C)
03/10/2018 (20.1°C)
10/10/2018 (19.7°C)
17/10/2018 (19.8°C)
24/10/2018 (20.8°C)
pH:
[C01; C02; C03]:
03/10/2018 : Control (C01) = 7.8 ; Solvent Control (DMSO) (C02) = 8.0 ; 2 mg.L-1 (C03) = 7.9 10/10/2018 : Control (C01) =7.9 ; Solvent Control (DMSO) (C02) = 8.0 ; 2 mg.L-1 (C03) = 8.0 17/10/2018: Control (C01) =7.9 ; Solvent Control (DMSO) (C02) = 7.9 ; 2 mg.L-1 (C03) = 7.9 24/10/2018: Control (C01) =7.8 ; Solvent Control (DMSO) (C02) = 7.9 ; 2 mg.L-1 (C03) = 7.9
Dissolved oxygen:
Dissolved Oxygen (mg.L-1) [C01 C02 C03]:
03/10/2018: Control (C01) = 9.1 ; Solvent Control (DMSO) (C02) = 9.0 ; 2 mg.L-1 (C03) = 8.7 10/10/2018: Control (C01) = 8.9 ; Solvent Control (DMSO) (C02) = 8.2 ; 2 mg.L-1 (C03) = 8.3 17/10/2018: Control (C01) = 8.1 ; Solvent Control (DMSO) (C02) =8.2 ; 2 mg.L-1 (C03) = 8.2 24/10/2018: Control (C01) = 8.4 ; Solvent Control (DMSO) (C02) = 8.0 ; 2 mg.L-1 (C03) = 8.3
Nominal and measured concentrations:
Limit test concentration: 2 mg/L in nominal concentration
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquarium
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass aquarium of 1.5 liters, filled to 1000 mL
- Aeration: The test vessel was aerated during the test.
- Type of flow-through (e.g. peristaltic or proportional diluter): The test was performed under continuous flow-through condition
- Renewal rate of test solution (frequency/flow rate):Test solution was prepared by dilution
of this solution in the OECD test medium, every day.
- No. of organisms per vessel:10 daphnia
- No. of vessels per concentration (replicates):4 replicates
- No. of vessels per control (replicates):4 replicates
- No. of vessels per vehicle control (replicates):4 replicates


TEST MEDIUM / WATER PARAMETERS
The test medium used was the OECD test medium (Elendt M4). It was reconstituted with chemical reagents and deionised water.The test medium was performed several times a week.

OTHER TEST CONDITIONS
- Photoperiod: A light:dark cycle of 16 hours light and 8 hours dark.
- Light intensity: Between 1000 and 1500 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Range finding study: Test concentrations: 100 µg/L ; 500 µg/L ; 2000 µg/L
- Results used to determine the conditions for the definitive study: Based on the results of the non-GLP range-finding test, a test limit was performed at 2 mg of test item.L-1 in Elendt-M4 medium.
Reference substance (positive control):
yes
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 3.94 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
> 3.94 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
> 3.94 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
> 3.94 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}
Details on results:
The total number of living offspring produced per parent animal alive was calculated for each replicate. If a parent animal dead during the test in a replicate treated with the test item and if
that mortality follows a dose response function, the parental mortality was considered as an
effect of the test item and these replicates were not excluded from the analysis. Otherwise,
replicates with parental mortality were be excluded from the analysis.
The LOEC, the NOEC and the ECx were determined using the following data:
-The total number of living offspring per parent animal not accidentally or fortuitously dead at
the end of the test.
-The total number of living offspring per parent animal alive at the end of the test.
The NOEC (No Observed Effect Concentration) were determined by statistical test from
replicate data on survival and reproduction.
Results with reference substance (positive control):
Results with reference substance were valid:
- Relevant effect levels: 24-hour EC50 at 1.85 mg/L with 95% confidence limits (mg/L) between 1.753 – 1.948
- Reference value : >= 0.6 mg/L and <= 2.1 mg/L.
- Compliance : Yes

Reported statistics and error estimates:
The NOEC (No Observed Effect Concentration) were determined by statistical test from
replicate data on survival and reproduction. Statistical calculations were made using R
statistical software.

On Reproduction:
A Shapiro-Wilk test was used to assess data normality and Bartlett’s test for homogeneity of
variance:

Shapiro-Wilk results:
W : Control (C01) = 0.9258; Solvent Control (DMSO)(C02) = 0.8930; 2 mg.L-1(C03) = 0.8712
p-value : Control (C01) = 0.5701 ; Solvent Control (DMSO)(C02) = 0.3970; 2 mg.L-1(C03) = 0.3024
p-value > 0.05: data normality is met for all conditions.

Bartlett’s test results:
Bartlett's K-squared = 0.2101, df = 2, p-value = 0.9003
p-value > 0.05: homogeneity of variance is met.

To check if a significant difference exists between concentrations and control, a t-test was
performed: p-value > 0.05 No significant difference.

On Survival:

A Shapiro-Wilk test was used to assess data normality and Kruskal-Wallis test (no parametric
test) to check if a significant difference exists between concentrations and control:

Shapiro-Wilk results:
W : Control (C01) = 0.6298 ; Solvent Control (DMSO)(C02) = 0.6298 ; 2 mg.L-1(C03) = 0.7286
p-value : Control (C01) = 0.001241 ; Solvent Control (DMSO)(C02) = 0.001241 ; 2 mg.L-1(C03) = 0.023857
p-value < 0.05: data normality is not met for all conditions.

Kruskal-Wallis results:
Kruskal-Wallis chi-squared = 0.6875, df = 2, p-value = 0.7091
p-value > 0.05: No significant difference.

Range finding test:

Sixty juveniles per adult were not reached either in control and tested concentrations as indicated in the OECD 211 recommendations. Indeed, we observed that a part of adults used were male that directly affected the total juveline production along the test.Despite this result, this range finding test allows to conlude that the substance has no significant effet on Daphnia magna reproduction at the highest tested concentration.

So a definitive limit test at 2 mg.L-1 was performed.

Geometric mean calculation:

At least three concentrations in a geometric series spaced by a constant spacing factor enables the choice of the appropriate limit concentration and the geometric means in μg test item/L were calculated. At a concentration of 100 μg test item/L the geometric mean was 10.04 μg test item/L , at 500 μg test item/ L the geometric mean was calculated to be 111.28 μg test item/L and at the highest concentration 2000 μg test item/L the geometric mean was 187.20 μg test item /L.

A significant deviation from the theoretical concentration was observed due to a decrease in the substance within 2 days before each medium renewal and the definitive test was performed under continual medium renewal.

The test substance concentration is not stable (i.e. outside the range 80 - 120% of nominal measured initial concentration), consideration was done for frequent medium renewal, so the test was performed under continuous flow-through condition.

A definitive limit test at 2 mg/L was then performed:

A stock solution in dimethyl sulfoxide (DMSO) was prepared at 50 g test item.L-1. Test solution was prepared by dilution of this solution in the OECD test medium, every day 


Validity criteria:

   Experimental value  Reference value     Compliance
 Mortality in control 0 -10 %   <  20%  Yes
 Mean number of living offspring produced per adult > 100   >=  60%  Yes
EC5024h potassium dichromate   1.85 mg/L

 >=

<=

0.6 mg/L 

2.1 mg/L

 Yes

The validity criteria are met.


Analytical results:

 Date Mesured concentration  (mg/L)     
   C01  C02  C03
 03/10/2018  0  0  3.1
 04/10/2018  0  0  3.26
 05/10/2018  <0.001 <0.001  3.61 
08/10/2018   0 6.02 
 09/10/2018  0  0 4.73 
10/10/2018   0  4.53
12/10/2018   0.00055  0.0007  4.70
 15/10/2018 2.99 
18/10/2018   0 0.00   3.93
22/10/2018   0 0.00   3.18
 24/10/2018  0 0.00   3.34
 Arithmetic Average 0.00   0.00  3.94

All results and subsequent calculations were based on arithmetic mean of measured concentrations.

Validity criteria fulfilled:
yes
Conclusions:
The objective of this study was to assess the effects of the test item on the reproductive output
of Daphnia magna over a period of 21 days according to OECD 211 guideline (2 october 2012).
Daphnia magna is a freshwater crustacean that is recommended by the OECD Guideline 211.
Since the test item is poorly soluble under the test conditions, a stock solution in dimethyl
sulfoxide was prepared at 20 g of test item.L-1. Test solution were prepared by dilution of this
solution in the OECD test medium.
Test item quantification was performed according to a validated analytical method (final report
RRCo-000154_01) using a LC-MS/MS from Sciex (LC Sciex Exion AD and MS QTRAP 5500)
as mass spectrometry detection to measure 2-(hexadecanoylamino)acetic acid
concentrations. As some of the measured concentrations were not within ± 20% of nominal or
initial concentrations, all results were based on mean measured concentrations.
The effects of the test item diluted in OECD test medium at the definitive measured
concentration of 2 mg.L-1 to Daphnia magna were investigated under laboratory conditions.

Results:
LC10 Survival (95% confidence limits) > 3.94 mg.L-1
LC50 Survival > 3.94 mg.L-1
EC10 Reproduction (95% confidence limits) > 3.94 mg.L-1
EC50 Reproduction > 3.94 mg.L-1
NOEC Survival > 3.94 mg.L-1
NOEC Reproduction > 3.94 mg.L-1
LC = lethal concentration ; EC = effect concentration

The study is valid since:
• the mortality of the parent animals (female Daphnia) was not exceed 20% at the end
of the test.
• the mean number of living offspring produced per parent animal surviving at the end
of the test was ≥ 60.
• EC50 with reference item over a period of 24 hours must be between 0.6 and 2.1 mg.L1

Based on the reproduction rate and the survival rate, the EC50 and LC50 after a 21 days
exposure period were greater than 3.94 mg.L-1. The NOEC was estimated to be at 3.94 mg.L-1
Executive summary:

The objective of this study was to assess the effects of the test item on the reproductive output of Daphnia magna over a period of 21 days according to OECD 211 guideline (2 october 2012). Daphnia magna is a freshwater crustacean that is recommended by the OECD Guideline 211. Since the test item is poorly soluble under the test conditions, a stock solution in dimethyl sulfoxide was prepared at 20 g of test item.L-1. Test solution were prepared by dilution of this solution in the OECD test medium.

Test item quantification was performed according to a validated analytical method (final report RRCo-000154_01) using a LC-MS/MS from Sciex (LC Sciex Exion AD and MS QTRAP 5500) as mass spectrometry detection to measure 2-(hexadecanoylamino)acetic acid concentrations. As some of the measured concentrations were not within ± 20% of nominal or initial concentrations, all results were based on mean measured concentrations.

The effects of the test item diluted in OECD test medium at the definitive measured concentration of 2 mg.L-1 to Daphnia magna were investigated under laboratory conditions. 


Based on the reproduction rate and the survival rate, the EC50 and LC50 after a 21 days

exposure period were greater than 3.94 mg.L-1. The NOEC was estimated to be at 3.94 mg.L-1


Description of key information

According to OECD 211 guideline (2 october 2012) to assess the effects of the test item on the reproductive output of Daphnia magna over a period of 21 days : Based on the reproduction rate and the survival rate, the EC50 and LC50 after a 21 days exposure period were greater than 3.94 mg.L-1. The NOEC was estimated to be at 3.94 mg.L-1

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
3.94 mg/L

Additional information