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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-04-13 to 2017-06-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline; EU Method; GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Reaction mass of disodium 4-{4-[acetyl(methyl)amino]-2-sulfonatoanilino}-1-amino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate and disodium sulfate and sodium chloride
EC Number:
944-710-7
Molecular formula:
not available because multi-constituent substance
IUPAC Name:
Reaction mass of disodium 4-{4-[acetyl(methyl)amino]-2-sulfonatoanilino}-1-amino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate and disodium sulfate and sodium chloride
Test material form:
solid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Duplicate samples from the freshly prepared test medium of the only test concentration and the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentration during the test period, duplicate samples from the test medium of the only test concentration and the control were collected at the end of the test (after 48 hours). All samples were diluted by factor 2 with Acetonitrile directly after sampling.
Additional samples of the untreated control and the dilution solvent were taken at test start and test end without any sample treatment.

Test solutions

Vehicle:
no
Details on test solutions:
Test Concentration:100 mg a.i./L (158.7 mg test item/L) and a control.
Control:In the control, test water was used without addition of the test item.
Dosage of Test Item:The test medium of the only test item concentration of nominal 100 mg a.i./L (158.7 mg test item/L) was prepared by dissolving 72.5 mg test item into 457 mL test water by intense stirring for 15 minutes. The test media was prepared just before introduction of the daphnids (= start of the test).

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 0.75 to 19.5 hours old
Sex: Female
Origin: The Daphnia introduced in the test were taken from ibacon's in house laboratory culture.
Breeding Conditions: The Daphnia were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental Daphnia was performed in Elendt M4 medium. The test organisms were not first brood progeny. The Daphnia in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
Water Hardness: 2.5 mmol/L (= 250 mg/L) as Calcium Carbonate
Test temperature:
18.9 to 19.0 °C at test start; 19.7 to 19.8 °C at test end
pH:
8.5 at test start;
7.7 at test end; and thus the pH-value did not vary by more than 1.5 units
Dissolved oxygen:
8.9 to 9.1 mg/L at test start;
8.9 mg/L at test end
Nominal and measured concentrations:
100 mg a.i./L (158.7 mg test item/L)
Details on test conditions:
Test Environment: Controlled environment room
Measurement of pH, Dissolved
Oxygen and Water Temperature:
The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.
Water Temperature: 18.9 to 19.0 °C at test start;
19.7 to 19.8 °C at test end
pH-Values: 8.5 at test start;
7.7 at test end; and thus the pH-value did not vary by more than 1.5 units
Dissolved Oxygen Concentration: 8.9 to 9.1 mg/L at test start;
8.9 mg/L at test end
Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 380 to 720 lux (measured once during the test).
Reference substance (positive control):
yes
Remarks:
For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Details on results:
After 48 hours of exposure no immobilisation of the test animals was observed in the control and in the only test concentration of nominal 100 mg a.i./L (158.7 mg test item/L).
Results with reference substance (positive control):
In the most recent test with the reference item potassium dichromate performed in January 2017 the EC50 after 24 hours was determined to be 1.48 mg test item/L, indicating that the sensitivity of the daphnids was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).
Reported statistics and error estimates:
No statistical analysis was performed.
The EC50 could not be quantified due to the absence of toxicity of the test item. The NOEC and the LOEC were determined directly from the raw data.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Control Immobilisation Rate:Was 0 %, no daphnid showed signs of disease or stress. Dissolved Oxygen Concentration:Was 8.9 mg O2/L in the control and test vessels at the end of the test; thus both validity criteria were met.
Conclusions:
The toxic effect of the test item to Daphnia magna was assessed in a static limit test. The 48-hour NOEC was determined to be at least 100 mg a.i./L (158.7 mg test item/L). The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg a.i./L (158.7 mg test item/L) have not been tested according to the recommendations of the test guidelines. The 48-hour LOEC and the 48-hour EC50 were clearly higher than 100 mg a.i./L (158.7 mg test item/L). These values could not be quantified due to the absence of toxicity of the test item up to 100 mg a.i./L (158.7 mg test item/L).
The quantification of the test item in the test samples was performed using liquid chromatography with UV detection.
At the start of the test 84% of the nominal test item concentration of 158.7 mg/L was found. After 48 hours test duration, 77% of the nominal value was determined. During the test the daphnids were exposed to a mean of 81% of nominal. Therefore, all reported results refer to the nominal concentrations.
Executive summary:

Title: test item: Acute Toxicity to Daphnia magna in a Static 48-hour Immobilisation Limit Test

Purpose: The purpose of this study was to determine the influence of the test item on the mobility of Daphnia magna. For this purpose, young daphnids (< 24 hours old) were exposed in a static test for 48 hours to the only concentration of nominal 100 mg a.i./L (158.7 mg test item/L) under defined conditions for 48 hours. The recorded effects were the mobility of the daphnids after 24 and 48 hours. This limit-test was performed in compliance with the test guidelines in order to demonstrate that the test item has no toxic effects on the daphnids up to this concentration. The test method of application and the test system are recommended by the test guidelines and Daphnia magna was the recommended test species. The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium.

Guidelines:

Commission Regulation (EC) No 440/2008, Annex, Part C, C.2.: “Daphnia sp. Acute Immobilisation Test”, Official Journal of the European Union (EN), dated May 30, 2008

OECD Guideline for Testing of Chemicals No. 202: “Daphnia sp., Acute Immobilisation Test” adopted April 13, 2004

OECD Series on Testing and Assessment, No. 23, “Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures”, December 15, 2000

SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

Material and Methods

Test Item; batch no.: CHA0 122490; purity: Acid Blue 182 (A. B. 182): 63.0% (w/w), according to certificate of analysis

Test Species: Daphnia magna, clone 5; 0.75 to 19.5 hours old

Source: The daphnids introduced in the test were taken from ibacon’s in-house laboratory culture.

Test Design: This study encompassed two treatment groups (one test item concentration at nominal 100 mg a.i./L (158.7 mg test item/L), and one control) each containing 20 individuals. The mobility of the daphnids was determined in a static 48-hour test by visual observation after 24 and 48 hours.

Endpoints: Number of immobile organisms after 24 and 48 hours

Test Concentrations: 100 mg a.i./L (158.7 mg test item/L) and a control.

Test Conditions: Water temperature: 18.9 to 19.8 °C; pH value: 7.7 to 8.5; dissolved oxygen concentration: 8.9 to 9.1 mg/L; photoperiod: 16 h light – 8 h dark; light intensity: 380 to 720 lux; and thus were within the ranges requested by guideline OECD 202

Biological test results: After 48 hours of exposure no immobilisation of the test animals was observed in the control and in the only test concentration of nominal 100 mg a.i./L (158.7 mg test item/L).

Conclusion: The toxic effect of the test item to Daphnia magna was assessed in a static limit test. The 48-hour NOEC was determined to be at least 100 mg a.i./L (158.7 mg test item/L). The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg a.i./L (158.7 mg test item/L) have not been tested according to the recommendations of the test guidelines. The 48-hour LOEC and the 48-hour EC50 were clearly higher than 100 mg a.i./L (158.7 mg test item/L). These values could not be quantified due to the absence of toxicity of the test item up to 100 mg a.i./L (158.7 mg test item/L). The quantification of the test item in the test samples was performed using liquid chromatography with UV detection. At the start of the test 84% of the nominal test item concentration of 158.7 mg/L was found. After 48 hours test duration, 77% of the nominal value was determined. During the test the daphnids were exposed to a mean of 81% of nominal. Therefore, all reported results refer to the nominal concentrations.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.