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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-11-20 to 2013-01-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study. Minor deviation from study plan did not affect the validty of the results.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
No media observations were made at 24 hours, but media observations consistent between 0 and 48 hours so did not impact validity of the results
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
see above
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Verification of test concentrations: Water samples from the control and 100 mg/L test group were taken at 0 (from the bulk test preparation) and 48 hours (replicates r1 - R4 pooled) for quantitative analysis.
Samples at 48 hours were frozen prior to analysis.
Duplicate samples were taken and frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Range-finding test
Nominal amounts of test item (10, 40 and 200 mg) were each separately added to the surface of 10, 4 and 2 litres of reconstituted water to give 1.0, 10 and 100 mg/L loading rates respectively. After the addition of the test item the 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 allowed to stand for 1 hour. 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 was removed by mid-depth siphoning (the first 75 - 100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rates. Microscopic observations of the test preparations showed there to be no micro-dispersions or undissolved test item present.

Definitive test
The test samples were prepared as above using 200 mg of the test material in 2L water to give the 100 mg/L loading rate.

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory culture, Harlan Laboratories.
- Age at study initiation (mean and range, SD): < 24 hours
- Breeding: parthogenesis
- Feeding during test: No

ACCLIMATION
- Acclimation period: not applicable culture maintained in-house. Adult Daphnia maintained in 150 mL glass beakers containing Elendt M7 medium. Gravid adults isolated day before initiation of test so young daphnids produced overnight were less than 24 hours old.
- Acclimation conditions (same as test or not): culture conditions same as test.
- Type and amount of food: Tetramin flake food suspension and algal suspension (Desmodesmus subspicatus)
- Feeding frequency: daily
- Health during acclimation (any mortality observed): not applicable culture maintained in-house. Adult Daphnia maintained in 150 mL glass beakers containing Elendt M7 medium. Gravid adults isolated day before initiation of test so young daphnids produced overnight were less than 24 hours old.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Not applicable
Hardness:
250 mg/L as CaCO3
Test temperature:
20 - 21°C
pH:
7.7 - 8.0
Dissolved oxygen:
8.7 - 8.9 mg O2/L
Salinity:
Not applicable
Nominal and measured concentrations:
Range finder: nominal loading rates of 1.0, 10 and 100 mg/L
Definitive test: 100 mg/L loading rate WAF
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass jar
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 200 mL test preparation
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours dark with a 20 minute dawn and dusk transition periods
- Light intensity: 955 - 990 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

Immobilisation or adverse reaction: 24 and 48 hours after start of exposure
Water temperature: daily
Dissolved oxygen content and pH: start and termination of test

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1.0, 10 and 100 mg/L loading rate WAF
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Loading rate WAF
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Loading rate WAF
Details on results:
Range-finding study
No immobilisation was observed at the 1.0, 10 and 100 mg/L loading rate WAF
Based on this information, a single loading rate of four replicates, of 100 mg/L was seleceted for the definitive test.
Definitive test
There was no immobilisation in 20 daphnids exposed to 100 mg/L loading rate WAF for a period of 48 hours. Insection ofthe immobilisation data gave the following results:
EL*50 (24 h) > 100 mg/L loading rate WAF
EL*50 (48 h) > 100 mg/L loading rate WAF
The No observed effect loading rate (NOELR) after 24 and 48 hours exposure was 100 mg/L loading rate WAF
Physico-chemical measurements
The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to th e presence of microscopic air bubbles in the media super-saturating the diluent and was not considered to have had an impact on the outcome or integrity of the study as no adverse effects were observed.
Test item solubility
At the start of the mixing period the 100 mg/L loading rate was observed to a be a clear colourless water column with oily globules of test item onthe surface. After 23 hours stirring and 1 hour standing the 100 mg/L loading rate was observed to remain a clear colourles water column with oily globules of test item onthe surface. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 100 mg/L loading rate was observed to be a clear colourless solution.
TOC analysis
TOC analysis of the fresh media at 0 hours showed a measured concentration of 2.55 mg C/L. Analysis of the old media at 48 hours showed a measured concentration of 0.39 mg C/L.
GIven that 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.
* EL - Efffective Loading Rate
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Mortality: yes
- EC50 (48 h) = 0.45 mg/L (95%CI = 0.42 - 0.48 mg/L)
- NOEC (48h) = 0.32 mg/L
Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item gave EL*50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.
Executive summary:

Introduction

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

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at single nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The number of immobilized Daphnia and any adverse reaction to exposure were recorded after 24 and 48 hours.

Results

Exposure of Daphnia magna to the test item gave EL*50 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.

Total Organic Carbon (TOC) analysis of the fresh media at 0 hours showed a measured concentration of 2.55 mgC/L. Analysis of the old media at 48 hours showed a measured concentration of 0.39 mg/L.

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.

*EL = Effective Loading Rate

Description of key information

Exposure of Daphnia magna to the test item gave EL*50 values 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

Additional information

A key 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.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at single nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The number of immobilized Daphnia and any adverse reaction to exposure were recorded after 24 and 48 hours.

Exposure of Daphnia magna to the test item gave EL*50 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.

Total Organic Carbon (TOC) analysis of the fresh media at 0 hours showed a measured concentration of 2.55 mgC/L. Analysis of the old media at 48 hours showed a measured concentration of 0.39 mg/L.

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.

*EL = Effective Loading Rate