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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:
12-14 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern guideline conform study performed in GLP certified laboratory

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Batch number: RW 2013-10-22
Color: Orange
Storage conditions: Room temperature
Expiring date: 22 October 2018

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
For determination of the test item concentrations, four replicates samples were taken from the test vessels of the treatment and the control groups at the start and at the end of the test, by using calibrated pipettes. A single concentration of 100 mg/L (limit concentration) and a concurrent control were included in the main test.

The analysis was performed by the analytical laboratory of TOXI-COOP ZRT according to the results of the analytical method validation (Study number: 805-100-4817) using reverse phase HPLC method.

Test solutions

Vehicle:
no
Details on test solutions:
The test solution was prepared by mechanical dispersion. An amount of 0.03 g test item was dissolved in 300 mL test medium (OECD medium) using approx. 15 min ultrasonic dispersion resulting in a concentration of 100 mg/L. The test solution was freshly prepared in the test laboratory just before introduction of the test animals (start of the experiment).

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Species and strain: Daphnia magna (Straus)
Origin: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection)
2100 Gödöllő, Kotlán S. u. 3. Hungary
Breeding: The Daphnia are bred in the Laboratory of TOXI-COOP ZRT.
Number of animals: Twenty animals in both groups (concentration and untreated control), divided into 4 concurrent batches (5 animals per batch).
Age of animals: Less than 24 h old at the beginning of the test.
Sex: Female
Animal health: Apparently healthy animals were used in this test with a known history
Acclimatization: Test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation before the test was not necessary. Brood daphnids were maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.
Food and Feeding: The Daphnia were fed with concentrated algal suspension of Pseudokirchneriella subcapitata during the holding. Test animals were not fed during the exposure.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
no

Test conditions

Hardness:
The hardness was determined to be 41.5 mg/L (as CaCO3) which (due to the chemical properties of the used test medium) is out of the recommended range of 140 - 250 mg/L, however there was no effect observed in behaviour of daphnids and therefore this value was considered to be acceptable and does not affect the results of the test. The hardness was measured using the AQUARAPID® – 1 Reagent set for determination of total hardness“ (Batch Number: KTB8752100, Supplier: REANAL, Expiry date: July 2014).
Test temperature:
The test temperature was in the range of 19.5 – 19.9 °C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 19.0 – 20.6 °C.
pH:
The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was in the range of 7.42 – 7.97 during the test.
Dissolved oxygen:
The dissolved oxygen concentration was in the range of 7.36 – 8.48 mg/L during the test.
Salinity:
N/A
Nominal and measured concentrations:
Test Item concentration measured on 12 March 2014:
Nominal concentration Mean of the measured concentrations Measured concentration in % of the nominal
mg/L (mg/L) with the 95% confidence interval (n=4) in % of the nominal
100 98.20 (RSD: 2 %) 98
Control not detected -

Table 4: Test Item concentration measured on 14 March 2014:
Nominal concentration Mean of the measured concentrations Measured concentration in % of the nominal
mg/L (mg/L) with the 95% confidence interval (n=4) in % of the nominal
100 99.25 (RSD: 1 %) 99
Control not detected -
Details on test conditions:
Composition of OECD medium
Stock solution Substance Final concentration in the prepared growth medium
Stock solution 1 (macro nutrients) NH4Cl 15.0 mg/L
Batch No.: PP/2012/09407
Supplier: REANAL (lach:ner)
Expiry date: 31 March 2017
MgCl2 (anhydrous) 5.6 mg/L*
Batch No.: 120M0094V
Supplier: SIGMA-ALDRICH
Expiry date: January 2015
CaCl2 × 2 H2O 18.0 mg/L
Batch No.: PP/2012/07312
Supplier: REANAL (lach:ner)
Expiry date: 30 November 2014
MgSO4 × 7 H2O 15.0 mg/L
Batch No.: PP/2011/05896
Supplier: REANAL (lach:ner)
Expiry date: 30 June 2015
KH2PO4 1.6 mg/L
Batch No.: AM0444073
Supplier: MERCK
Expiry date: 31 July 2017
Stock solution 2 (iron) FeCl3 × 6 H2O 64.0 g/L
Batch No.: BCBL0680V
Supplier: SIGMA-ALDRICH
Expiry date: 28 May 2016
Na2EDTA × 2 H2O 100.0 g/L
Batch No.: K44403118
Supplier: MERCK
Expiry date: 30 April 2018
* This corresponds to the concentration of 12.0 mg/L of MgCl2 × 6 H2O (cf. OECD no. 201 guideline)

Table 2(continued): Components of the OECD Medium
Stock solution Substance Final concentration in the prepared growth medium
Stock solution 3 (trace elements) H3BO3 185.0 g/L
Batch No.: A0292565
Supplier: MERCK
Expiry date: 30 April 2016
MnCl2 × 4 H2O 415.0 g/L
Batch No.: A0250927
Supplier: MERCK
Expiry date: 31 December 2015
ZnCl2 3.0 g/L
Batch No.: B0957416
Supplier: MERCK
Expiry date: 31 July 2015
CoCl2 × 6 H2O 1.5 g/L
Batch No.: B0396239
Supplier: MERCK
Expiry date: 30 April 2014
CuCl2 × 2 H2O 0.01 g/L
Batch No.: K43082433
Supplier: MERCK
Expiry date: 31 December 2016
Na2MoO4 × 2 H2O 7.0 g/L
Batch No.: SLBC3966V
Supplier: SIGMA-ALDRICH
Expiry date: May 2016
Stock solution 4 (bicarbonate) NaHCO3 50.0 mg/L
Batch No.: PP/2012/05232
Supplier: REANAL (lach:ner)
Expiry date: 31 January 2017

Test Conditions

The test vessels were kept during the test in a climate chamber under controlled conditions. The dissolved oxygen concentration and pH were measured in the test item treated and the control group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of the test. The water temperature was measured in each test vessel at the start and at the end of the test. Additionally, the ambient temperature was measured continuously using a min/max thermometer in the climate chamber.

Test type: Static test, based on the results obtained during analytical method validation (Study number: 805-100-4817). The test item was shown to be stable for 48 hours.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
A concentration of 100 mg/L (limit concentration) and a concurrent control was included in the main test. The concentration of the test item was analytically determined at the start and at the end of the experiment. The test item was not detected in the untreated control group. In the treated group measured concentrations of test item were 98 % of the nominal at the start and 99 % at the end of the test. The measured test item concentration remained within ± 20 % of the nominal during the test period of 48 hours, therefore biological results are given as nominal concentration.

Biological Results

There was no immobilisation observed in twenty daphnids exposed to each test item treated and control group. Further no abnormal behaviour or appearance of test animals was noted.
The 48-h NOEC was determined to be 100 mg/L. The 48-h LOEC, EC100 and the EC50 values were determined to be > 100 mg/L.
Results with reference substance (positive control):
For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 392-202-4865) with reference item Potassium dichromate was: 05 – 06 March 2014.
The 24-h EC50: 1.31 mg/L, (95 % confidence limits: 1.15 – 1.60 mg/L)
Reported statistics and error estimates:
A limit test was performed and toxic effects were not observed, therefore statistical analysis was not necessary.
The NOEC, LOEC, EC50 and EC100 values of the test item were determined directly from the raw data.

Any other information on results incl. tables

Immobilization of the test animals

Test Group

Replicate

Number of
treated animals

Number of immobilised animals

24 h

48 h

Control

1

5

0

0

2

5

0

0

3

5

0

0

4

5

0

0

100 mg/L

1

5

0

0

2

5

0

0

3

5

0

0

4

5

0

0

 

Applicant's summary and conclusion

Conclusions:
In this 48-hour static acute toxicity test with Daphnia magna the test item YELLOW LF 6881 had no toxic effect on Daphnia up to a concentration of 100 mg/L. Accordingly, the 48-h EC50 value was determined to be > 100 mg/L. The 48-h NOEC was determined to be 100 mg/L.
Executive summary:

The substance YELLOW LF 6881 was assessed usingDaphniamagnain a 48-hour Acute Immobilisation Test. YoungDaphniawere exposed to aqueous test media containing the test item for 48 hours.Based on results obtained in a preliminary experimentDaphniawere exposed to a single concentration of 100 mg/L (limit concentration). A concurrent control group was run.

A static test was performed as the test item was previously shown to be stable in the test medium for 48 hours (Toxi-Coop Study No:805-100-4817). The analytically measured test item concentration remained within ± 20 % of the nominal during the test period of 48 hours; therefore, biological results are based on the nominal concentration.

Healthy, young female daphnids with known origin, history and acclimatisation conditions were used. TwentyDaphnia(divided into 4 concurrent batches) were tested each, exposed to 100 mg/L test item and in the control. Each test vessel contained approximately 40 mL test medium.Fully characterized (content, physico-chemical characteristics) OECD Medium was used as test medium.

The immobilisation of the test animals was recorded 24 and 48 hours after treatment. Environmental conditions were recorded. All measured values remained within acceptable ranges.

All validity criteria were met. There was no immobilisation observed in the twenty daphnids exposed to either the test item or in the control group. In addition to immobility, no abnormal behaviour or appearance of test animals was noted.

The 48-h NOEC was determined to be 100 mg/L and the LOEC, EC100and the EC50values were determined to be > 100 mg/L. The obtained results are summarised in Table 1 below.

 

Table 1:Summary of the Biological Results

Parameter
[mg/L]

Time period

24 h

48 h

EC50

> 100

> 100

48 h-NOEC

100

48 h-LOEC

> 100

48 h-EC100

> 100