RD 14156

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 23 May 20016 and 24 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor Lot/Batch number: 5399561P10
- Expiration date of the lot/batch:25 January 2020
- Purity test date: 25 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item

Analytical monitoring:

yes

Details on sampling:

Based on the results of the range-finding test, the following loading rates were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.

Nominal amounts of test item (50, 90, 160, 280 and 500 mg) were each separately added to the surface of 5 liters of test water to give the 10, 18, 32, 56 and 100 mg/L loading rates, respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours, and the mixtures were allowed to settle for 1 hour. Observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column, and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10, 18, 32, 56 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no microdispersions of test item present.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24 (fresh and old) and 48 hours

Vehicle:

no

Details on test solutions:

Nominal amounts of test item (50, 90, 160, 280 and 500 mg) were each separately added to the surface of 5 liters of test water to give the 10, 18, 32, 56 and 100 mg/L loading rates, respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours, and the mixtures were allowed to settle for 1 hour. Observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column, and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10, 18, 32, 56 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no microdispersions of test item present

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Annex 2) in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

22-23”C

pH:

7.8-8.2

Dissolved oxygen:

8.1-8.6 mgO2/L

Nominal and measured concentrations:

Chemical analysis of the freshly prepared 100 mg/L loading rate test preparations at 0 and 24 hours showed measured test concentrations of 0.20 and 0.16 mg/L, respectively. Chemical analysis of the aged 100 mg/L loading rate test preparations at 24 and 48 hours showed measured test concentrations of 0.14 and 0.099 mg/L, respectively

Details on test conditions:

250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test, 5 daphnids were placed in each test concentration and control vessel at random. Four replicate test concentration and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ± 1 °C with a photoperiod of 16 hours light (between 200 and 1200 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality

Details on results:

There was no immobilization in 20 daphnids exposed to the test concentration series of 10, 18, 32, 56 and 100 mg/L Loading Rate WAF for a period of 48 hours.
The No Observed Effect Loading Rate after 24 and 48 hours of exposure was 100 mg/L loading rate WAF.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Results with reference substance (positive control):

A positive control (Envigo Study Number MS29MC) used potassium dichromate as the reference item at test concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
The ToxRat Professional computer software package was used for analysis of the immobilization data at 24 and 48 hours by the probit analysis using the linear maximum likelihood regression method
The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No. 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 22 to 23 C under semi-static test conditions. The number of immobilized daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Chemical analysis of the fresh 100 mg/L loading rate WAF test preparations at 0 and 24 hours showed measured test concentrations of 0.20 mg/L and 0.16 mg/L, respectively. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations of 0.14 and 0.099 mg/L, respectively.

The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Daphnia magna to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

Description of key information

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL50 value of greater than 100 mg/L loading rate WAF.  The No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L