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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:
12 April 2010 and 28 June 2010.
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)
Remarks:
Date of GLP inspection:15 september 2009 Date of Signature on GLP certificate: 26 November 2009
Analytical monitoring:
yes
Details on sampling:
The test concentration 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 test concentrations of 10 and 100 mg/l. The test item was dissolved directly in water.

Verification of test concentrations
Water samples were taken from the control and each replicate test vessel at 0 (fresh media), 24 and 96 (old media) hours for quantitative analysis.
Duplicate samples and samples at 24 (fresh media), 48 and 72 hours (fresh and old media) were taken and stored at approximately -20ºC for further analysis if necessary.
The method of analysis, stability, recovery and test preparation analyses are described in Appendix 2 see in attached section.
Vehicle:
no
Details on test solutions:
An amount of test item (4000 mg) was dissolved in dechlorinated tap water and the volume adjusted to 2 litres to give a 2000 mg/l stock solution. Aliquots (100 and 1000 ml) of this stock solution were separately added to a final volume of 20 litres of dechlorinated tap water to give the 10 and 100 mg/l test concentrations respectively.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test fish were added to each 20 litre test and control vessel and maintained at approximately 14ºC in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours under static test conditions.
The control group was maintained under identical conditions but not exposed to the test item.
Data from the control group was shared with similar concurrent studies.
Each vessel was covered to reduce evaporation. 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.

Definitive test
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/l to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines no mortalities or sub-lethal effects of exposure were observed.

Experimental Preparation
For the purpose of the definitive test the test item was dissolved directly in water.
An amount of test item (4000 mg) was dissolved in dechlorinated tap water and the volume adjusted to 2 litres to give a 2000 mg/l stock solution. Aliquots (1000 ml) of this stock solution were each added separately to a final volume of 20 litres of dechlorinated tap water and stirred with a flat bladed mixer for approximately 1 minute to give the 100 mg/l test concentrations Replicates 1 and 2.
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 obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 12 May 2010. Fish were maintained in a glass fibre tank with a "single pass" water renewal system. Fish were acclimatised to test conditions from 9 June 2010 to 21 June 2010. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

The water temperature was controlled at approximately 14ºC with a dissolved oxygen content of greater than or equal to 10.1 mg O2/l. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test. There was 0 % mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.0 cm (sd = 0.4) and a mean weight of 1.58 g (sd = 0.40) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.55 g bodyweight/litre (static volume).
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Not applicable
Hardness:
Total hardness of approximately 140 mg/l as CaCO3.
Test temperature:
The test vessels were Covered and maintained at approximately 14ºC
pH:
The pH was measured using a WTW pH/Oxi 340I pH.
pH range of 7.5-8.5.
Please see Physico-Chemical Measurements appendix 3 (see in any other information on materials and methods).
Dissolved oxygen:
The dissolved oxygen concentration was measured using a dissolved oxygen meter.
Please see Physico-Chemical Measurements appendix 3 (see in any other information on materials and methods section).
Salinity:
Not applicable
Nominal and measured concentrations:
10 and 100 mg/L
Details on test conditions:
Exposure conditions
As in the range-finding test 20 litre glass exposure vessels were used for each test concentration. At the start of the test 7 fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at approximately 14ºC in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.
The control group was maintained under identical conditions but not exposed to the test item. Data from the control group was shared with similar concurrent studies.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure that the concentrations of the test item remained near nominal and to prevent the build up of nitrogenous waste products.
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

Physico-chemical measurements
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The pH was and the dissolved oxygen concentration was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter and the temperature was measured using Hanna Instruments HI 93510 digital thermometer.


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 (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) 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. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 1 see attached section.



Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL Not stated
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL Not stated
Details on results:
Range-finding Test
Cumulative mortality data from the exposure of rainbow trout to the test item during the range-finding test are given in Table 1 see in any other information in results section There were no sub-lethal effects of exposure during the range-finding test.
A slightly cloudy water column was observed after 72 hours in the 100 mg/l test concentration which was considered to be precipitation of the test item. However no adverse effects were observed and given that the definitive test was to be conducted under semi-static test conditions with a renewal period of 24 hours, it was considered that this precipitation would have no effect on the outcome or validity of the test.
Based on this information, a single test concentration, in duplicate, of 100 mg/l was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EEC Test Guidelines no mortalities or sub lethal effects of exposure were observed.

Definitive Test
Mortality data
Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 2 see in any other information in results section.
There were no mortalities in 14 fish exposed to a test concentration of 100 mg/l for a period of 96 hours. Inspection of the mortality data gave the following results:
Time (h) LC50 (mg/l)
3 > 100
6 > 100
24 > 100
48 > 100
72 > 100
96 > 100
The results of the definitive test showed the No Observed Effect Concentration (NOEC) to be 100 mg/l. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.

Sub-lethal effects
There were no sub-lethal effects of exposure observed in 14 fish exposed to a test concentration of 100 mg/l for a period of 96 hours.

Observations on test item solubility
The test media was observed to be a clear colourless solution throughout the duration of the test.

Physico-chemical measurements
The results of the physico-chemical measurements are given in Appendix 3 see in any other information in materials and method section. Temperature was maintained at approximately 14ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.
The pH of the control group was observed to vary between 7.5 and 8.5. This variation was considered not to affect the validity or integrity of the test given that no mortalities or adverse reactions to exposure were observed in the control group and the Test Guideline states that the pH should not vary by more than 1 unit.

Verification of test concentrations
Analysis of the test preparations at 0, 24 and 96 hours (see Appendix 2 in attached section) showed measured test concentrations to range from 100% to 110% of nominal and so it was considered justifiable to estimate the LC50 values in terms of the nominal test
Results with reference substance (positive control):

- Results with reference substance valid
Not applicable

- Mortality:
Not applicable

- LC50:
Not applicable

- Other:
Not applicable

Table 1              Cumulative Mortality Data in the Range-finding Test

Nominal

Concentration

(mg/l)

Cumulative Mortality

(Initial Population = 3)

3 Hours

6 Hours

24 Hours

48 Hours

72 Hours

96 Hours

Control

0

0

0

0

0

0

10

0

0

0

0

0

0

100

0

0

0

0

0

0

Table2              Cumulative Mortality Data in the DefinitiveTest

Nominal

Concentration

(mg/l)

Cumulative Mortality
(Initial Population = 7)

%

Mortality

3 Hours

6 Hours

24 Hours

48 Hours

72 Hours

96 Hours

96 Hours

Control

0

0

0

0

0

0

0

100 R1

0

0

0

0

0

0

0

100 R2

0

0

0

0

0

0

0


R1and R2= Replicates 1 and 2

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 LC50 of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.
This study has been selected as the key study because the results are sufficient in order to derive a reliable conclusion on classification and labelling in accordance with Regulation EC (No.) 1272/2008 (EU CLP).
Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed that described in 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.

Following a preliminary range-finding test fish were exposed, in two groups of seven, to an aqueous solution of the test item, at a single concentration of 100 mg/l for a period of 96 hours at a temperature of approximately 14ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results.

The 96-Hour LC50based on nominal test concentrations was greater than 100 mg/l and correspondingly the No Observed Effect Concentration was 100 mg/l.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.

Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 100% to 110% of nominal and so the results are based on nominal test concentrations only.

Conclusion.

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.

Description of key information

A key study to assess the short-term toxicity of sodium metaphosphate to fish has been performed according to OECD guideline 203 and under the conditions of GLP.

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 of greater than 100 mg/l.  Correspondingly the No Observed Effect Concentration was 100 mg/l.