Formaldehyde, reaction products with 5,12-dihydroquino[2,3-b]acridine-7,14-dione and 3,5-dimethyl-1H-pyrazole, sulfonated

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:

2007-12-18 to 2008-05-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline Study (OECD 202) including analytical monitoring

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: JMAFF Test Guidelines, 2-7-2-1, Daphnia immobilization, 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: limit test concentration and control
- Sampling method: For the determination of the actual test item concentrations, duplicate samples were taken from each treatment before the start of the test and at the end of the test after 48 hours.
For the 48-hour stability samples additional flasks with adequate volumes of the freshly prepared test medium and of the control were incubated during the test period under the same conditions as in the actual test (but without daphnids). Sampling from the test beakers with daphnids was not possible because the test medium volume per beaker (50 mL) was too small for the analytical requirements (400 mL per sample).
- Sample storage conditions before analysis: All samples were stored deep-frozen (at about -20 °C) immediately after sampling. Based on pre-experiments for investigation of the storage stability (without GLP), the test item was found to be sufficiently stable in the test water under these storage conditions.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method:
A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the daphnids up to the loading rate of 100 mg/L. Thus, a single loading rate of 100 mg/L was tested.

- Controls:
Additionally, a control was tested in parallel (test water without addition of the test item). The limit test was based on the results of a range-finding test and on results of a pre-experiment to determine the solubility of the test item in the test water.

- Test medium:
For the preparation of the test medium, a dispersion of the test item with the loading rate of 100 mg/L was prepared. The dispersion was stirred for 3 hours to dissolve a maximum amount of the different compounds of the test item in the dispersion. Then, the dispersion was filtered through a membrane filter (0.45 μm pore size) and the undiluted filtrate was tested as WAF.

- Standard Solutions for Sample Quantification:
The test item (20.14 mg) was dissolved in dimethylacetamid, made up to the mark in a 100 mL volumetric flask and sonicated for 10 minutes to prepare a stock solution of 201 mg/L. Defined volumes of this stock solution were diluted with dimethylacetamid to obtain standard solutions of the test item in the range of 0.0504 to 20.1 mg/L.
These solutions were used to calibrate the HPLC-system.

- Preparation of Spiked Test Water Samples:
The test item (22.24 mg) was dissolved in dimethylacetamid, made up to the mark in a 100 mL volumetric flask and sonicated for 10 minutes to prepare a stock solution of 222 mg/L. 1 mL of this stock solution was made up to 100 mL with dimethylacetamid to obtain a solution with a concentration of 2.22 mg/L of the test item. 0.5 mL of this solution was diluted with test water to obtain spiked samples with a concentration of 0.00278 mg/L. This solution was freshly prepared in duplicate and subjected to the same treatment as a sample without any storage.
In addition, test water without the test item was analyzed (analytical blank).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- The study was performed with young daphnids of the species Daphnia magna Straus.
- Source: A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992. Since that time, the clone has been bred in the laboratories of RCC in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
- Age at study initiation: At the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

250 mg/L as CaCO3

Test temperature:

20 °C

pH:

7.7 - 7.8

Dissolved oxygen:

8.0 - 8.4 mg/L

Salinity:

Alkalinity: 0.8 mmol/L;

Nominal and measured concentrations:

Loading rate: 100 mg/L;
Measured concnetration: 0.000161 mg/L;

Details on test conditions:

TEST SYSTEM
A static test without test medium renewal was performed.
- Test vessel: The test was performed in 100-mL glass beakers filled with 50 mL of test medium.
- Type: The test vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.
The test was performed in a temperature-controlled room with continuous monitoring of the room temperature.

TEST MEDIUM / WATER PARAMETERS
- Photoperiod: A 16-hour light to 8-hour dark cycle with a 30 minute transition period was used.
- Light intensity: Light intensity during the light period was between approximately 490 and 650 Lux.
- The daphnids were not fed during the test.
- No. of organisms per vessel: For each treatment, 20 daphnids were used divided into four replicates of five daphnids each.
- The volume of test solution provided for each daphnia was 10 mL.
- The daphnids were randomly distributed to the test vessels at initiation of the test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: No acute toxic effects within the range of solubility.

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: No acute toxic effects within the range of solubility.

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: EC50 was estimated; no acute toxic effects within the range of solubility.

Details on results:

A limit test was performed in accordance with the test guidelines to demonstrate that water accommodated fractions (WAFs) of the test item have no toxic effect on Daphnia magna up to the loading rate of 100 mg/L. Thus, the only concentration tested was a WAF with the loading rate of 100 mg/L in parallel to a control.
In the control and at the loading rate of 100 mg/L, no immobilized daphnids were determined during the test period of 48 hours.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

With high probability not acutely harmful to aquatic invertebrates. No acute toxic effects within the range of solubility.