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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 17 September 2013 and 17 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Description: Clear, colourless liquid
Batch: 20130510
Purity: not applicable - complex mixture
Expiry date: 10 May 2018
Storage conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and the 100 mg/L loading rate WAF test vessel at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) for quantitative analysis. The samples were frozen prior to analysis.

Duplicate samples, and samples at 24 (fresh media), 48 and 72 hours (old and fresh media), were taken and stored frozen for further analysis if necessary.

Vehicle:
no
Details on test solutions:
An amount of test item (2100 mg) was added to the surface of 21 litres of test water to give the 100 mg/L loading rate. 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 71 hours and the mixture allowed to stand for 2 hours. Visual observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel. A length of Tygon tubing was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic observation of the WAF were performed after filtering and showed that the filtration had removed all of the undissolved test item present in the water column.

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 26 September 2013 to 8 October 2013.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
140 mg/L CaCO3
Test temperature:
13 to 14 ºC
pH:
7.7 to 8.4
Dissolved oxygen:
10.2 to 10.5 mg O2/L
Salinity:
Not measured
Conductivity:
Not measured
Nominal and measured concentrations:
Loading rate of 100 mg/L
Details on test conditions:
The water temperatre was controlled at approximately 14 ºC with a dissolved oxygen content of greater than or equal to 9.0 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.1 cm and a mean weight of 1.30 g at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.46 g bodyweight/L.

The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.

Laboratory tap water was dechlorinated by passage through an activated carbon filter and partly softened giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

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.

Range finding test:
the loading rate to be used in the definitive test was determined by a preliminary range-finding test. In the range finding test fish were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L. After 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish. The results showed no mortalities at 1.0, 10 and 100 mg/L lading rate WAF. Based on this information, a single loading rate of 100 mg/L was selected for the definitive test.

Definitive test:
Based on the results of the range-finding test a Limit test was conducted at a single loading rate of 100 mg/L to confirm that no mortaliites or sub-lethal effects of exposure were observed. At the start of the test 7 fish were placed in each test vessel at random, in the test preparations.

Any mortalities and sub-lethal effects of exposure were recorded at 3, 24, 48, 72 and 96 hours after the start of the exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
3 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
24 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
72 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
There were no mortalities in 7 fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours.
There were no sub-lethal effects of exposure observed in 7 fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours.
Results with reference substance (positive control):
N/A
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-hour LL50 value of >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 rainbow trout. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 203 "Fish, Acute Toxicity Test" referenced as Method C. 1 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, seven fish were exposed to a WAF of the test item, at a single, nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of approximately 13 ºC under semi-static conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results

Chemical analysis of the freshly prepared 100 mg/L loading rate WAF at 0 and 72 hours showed that measured concentrations of 0.085 and 0.18 mg/L were obtained respectively. Analysis of the old or expired media at 24 and 96 hours showed that measured concetration of 0.033 and 0.14 mg/L were obtained respectively.

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 rainbow trout to the test item gave LL50 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 fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-hour LL50 value of >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

LC50 for freshwater fish:
100 mg/L

Additional information