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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-10-02 to 2014-05-23
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2 october 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
Name of the test material(as cited in study report):Proteol APL
batches:
0824000008
0824200013
0824200014

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1209000017
- Purity: 31.7% (dry extract)
- Expiration date of the lot/batch: 2015-03-30
- Purity test date: 2012-04-17

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temparature



Sampling and analysis

Analytical monitoring:
yes
Remarks:
The whole concentration range was measured twice during the experiment before and after renewal and three test concentrations were measured throughout the test
Details on sampling:
Each sample before renewal has been centrifuged at 4000 g during 15 minutes. When samples were not analysed the day of sampling, they were frozen until analysis.

Test solutions

Vehicle:
yes
Remarks:
test medium (Elendt M4)
Details on test solutions:
The test item Sodium cocoyl apple amino acids is soluble in water. A stock solution of 200 mg/L was prepared in the test medium (Elendt M4) for each renewal. Different amounts of the stock solution were then diluted in the test medium (Elendt M4) to obtain the selected concentration. 50 mL of these solutions were introduced into the glass vessel before introduction of organisms.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna Straus 1820 (Crustacea Cladocera): Clone A, coming from a continuous and balanced breeding carried out in the test facility, aged less than 24 hours at the start of the test.

Study design

Water media type:
saltwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
not applicable

Test conditions

Hardness:
260 mg/L expressed as CaCO3
Test temperature:
between 19 and 21.5°C
pH:
8.1 – 8.2
Dissolved oxygen:
8.7 – 8.9 mg/L
Conductivity:
689 - 695 μS/cm
Nominal and measured concentrations:
1.00 – 2.41 - 5.79 - 13.9 - 33.3 – 80.0 mg/L (initial nominal concentrations)

Two reasons led to expressed the results in nominal concentrations :
1) The lack of quantification of the test item before the renewal of the test solutions
2) The measurement of part only of the test concentrations
Nevertheless, the initial measured concentrations remained within ± 20% of the nominal values of concentrations
Details on test conditions:
- Vessels: disposable glass bottles of 90 mL capacity filled with 50 mL of test solution
- Number of animals: 10 daphnids individually held at each test concentration and for the control
- Dilution water: Elendt M4

Measurements: - Mortality among the parent at each working day
- The offspring (living and death) produced by each parent animal at each working day
- pH and oxygen dissolved at each renewal
- Test concentrations: 1.00 – 2.41 – 5.79 – 13.9 – 33.3 – 80.0 mg/L (initial nominal concentrations). Test concentrations have been determined according to the results of an acute immobilisation test (48 h)
- Renewal of test solutions: 3 times per week
- Feeding: 26.0 μL of concentrated algae suspension (Chlorella vulgaris) equivalent to 0.15 mg C/Daphnia/day at each working day
- Observation: at each working days
- Aeration: none within the test period
- Environmental conditions: 1000 – 1500 lux, 16 hours light, temperature between 19 and 21.5°C
- Duration: 21 days
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
21 d
Dose descriptor:
NOEC
Remarks:
reproduction
Effect conc.:
ca. 33.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EC50
Remarks:
mortality
Effect conc.:
ca. 43.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
As a result of the lack of quantification of the test item after 48 hours of exposition, a new series of test flasks was added in the same test conditions from the 19th day of the experiment, but without organism and without algae in order to estimate the behaviour of the test item only in the test medium. Concentration measurements carried out on these flasks were similar to the results obtained for flasks with organisms and algae i.e. lower than the LQ.
The analytical system (LC MS/MS) used in this study is a very accurate detection system based on the molecular weight of the test item analysed. If the molecular weight of the test item is modified, for example, by cation exchange (as explained in the "Expert Notification" in Annex 8), the test item will not be detected by the analysis system.
This reaction could explain the non-detection of the test item, while the initial concentrations are satisfactory, after only 24 hours of exposure in the test medium.
Furthermore, a ready biodegradability test carried out in 20042, showed that only 22% of the test item was biodegraded after 48 hours (49% after 5 days) under the conditions of achievement of this test.
Consequently, the lack of analytical quantification of the test item after 24 or 48 hours in the test medium cannot be mainly linked to the biodegradation of the test item over such a short exposure period.
Results with reference substance (positive control):
The test carried out from 09 to 10 October 2013 (in NF EN ISO 6341) gave the following results:
EC50 24h: 1.08 mg/L (1.03 – 1.13 mg/L).
The EC50 24 hours was calculated using a logistic regression analysis with a bootstrap estimation of the 95% confidence limits.
The EC50 24h is in the recommended range: 0.6 – 2.1 mg/L. This result confirms the appropriate sensitivity of the strain of Daphnia magna.
Reported statistics and error estimates:
Determination of effect on parental survival
Statistical tests (Toxstat 3.5, Western Inc. and D. Gulley, University of Wyoming) were performed to determine the Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) in the test conditions.
As the assumption of the variance homogeneity was not fulfilled for the data obtained during this study, non-parametric tests were carried out (Steel’s Many-One Rank Test and Wilcoxon’s Rank Sum Test). These statistical tests weren’t able to accurately determine a LOEC and consequently the NOEC.

Determination of effect on the reproduction (offspring produced per surviving parents)
Statistical tests (Toxstat 3.5, Western Inc. and D. Gulley, University of Wyoming) were performed to determine the Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) in the test conditions.
As the assumption of the variance homogeneity was not fulfilled for the data obtained during this study, non-parametric tests were carried out (Steel’s Many-One Rank Test and Wilcoxon’s Rank Sum Test) which showed a statistically significant effect at the highest concentration tested for the definitive study, i.e. 80.0 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The results obtained during this study showed a concentration-effect relationship for the Sodium cocoyl apple amino acids on the Daphnia magna reproduction.
At the end of the exposure period, an EC50 for two criteria: mortality and reproduction was determined between 33.3 and 80.0 mg/L, a No Observated Effect Concentration was determined at 33.3 mg/L for the reproduction criterion and no mortality was observed at the test concentration of 13.9 mg/L (initial nominal concentration).
Two reasons led to expressed the results in nominal concentrations
1) The lack of quantification of the test item before the renewal of the test solutions
2) The measurement of part only of the test concentrations
Nevertheless, the initial measured concentrations remained within ± 20% of the nominal values of concentrations.
Executive summary:

TEST PURPOSE

Determination of the effect of the test item Sodium cocoyl apple amino acids on the reproductive output of a test population of Daphnia magna within a 21 days exposure period. The test is carried out according to the procedure described in the OECD guideline for testing of chemicals n°211 "Daphnia magna, Reproduction test"; in conformity with the clauses recommended in the study plan of 2 October 2013, signed by the sponsor on 4 October 2013.

GUIDELINE

OECD guideline for testing of chemicals n°211 "Daphnia magna Reproduction Test" (Adopted 2 October, 2012).

TEST SYSTEM

Daphnia magna

TEST ITEM

Sodium cocoyl apple amino acids

TEST CONCENTRATIONS

1.00 – 2.41 - 5.79 - 13.9 - 33.3 – 80.0 mg/L (initial nominal concentrations)

DURATION

21 days

RESULTS

Results expressed as initial nominal concentrations (mg/L):

 Test Item  NOEC (reproduction)  EC50 (reproduction) EC50 (survival)
 Sodium cocoyl apple amino acids  33.3 mg/L 33.3 < EC50 21d < 80.0 43.1 (34.1 – 52.6) 

CONCLUSION

The results obtained during this study showed a concentration-effect relationship for the Sodium cocoyl apple amino acids on the Daphnia magna reproduction. At the end of the exposure period, an EC50 for two criteria: mortality and reproduction was determined between 33.3 and 80.0 mg/L, a No Observated Effect Concentration was determined at 33.3 mg/L for the reproduction criterion and no mortality was observed at the test concentration of 13.9 mg/L (initial nominal concentration).

Two reasons led to expressed the results in nominal concentrations

1) The lack of quantification of the test item before the renewal of the test solutions

2) The measurement of part only of the test concentrations Nevertheless, the initial measured concentrations remained within ± 20% of the nominal values of concentrations.