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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:
24 February 2010 to 22 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
(see principles of method if other than guideline)
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
yes
Remarks:
(see principles of method if other than guideline)
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Previous experience gained from studies conducted on poorly water soluble test items has shown that a mixing period of 24 - 48 hours is sufficient to ensure equilibration between the test item and water phase. At the completion of mixing, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 15/09/2009 Date of Signature on GLP certificate: 26/11/09
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Nominal loading rates of 10, 18, 32, 56 and 100 mg/L

- Sampling method:
Water samples were taken from the control and all surviving test groups at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media) for quantitative analysis.

- Sample storage conditions before analysis:
Duplicate samples were taken and stored at approximately -20°C for further analysis, if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Rsults from short-term toxicity testing to aquatic invertebrates (Harlan Laboratories Ltd Project Number: 2702/0161) showed toxicity at loading rates of 56 and 100 mg/L. Therefore, the following loading rates were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.

- Method:
Due to the low aqueous solubility and complex nature of the test material, for the purposes of the definitive test, the test material was prepared as a Water Accommodated Fraction (WAF).

Amounts of test material (210, 378, 672, 1176 and 2100 mg) were each separately added to the surface of 21 litres of dechlorinated tap water to give the 10, 18, 32, 56 and 100 mg/L loading rates, respectively. After the addition of the test material, the dechlorinated tap 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 47 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test material 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. A glass wool plug 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 10, 18, 32, 56 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro-dispersions to be present.


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

- Controls:
Control groups were maintained under identical conditions but not exposed to the test material.

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow Trout
- Source: Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation (mean and range, SD): Juvenile
- Length at study initiation (length definition, mean, range and SD): Fish had a mean standard length of 5.2 cm (SD = 0.5)
- Weight at study initiation (mean and range, SD): Mean weight of 1.99 g (SD = 0.44) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.70 g bw/L
- Feeding during test: discontinued 24 hours prior to the start of the definitive test
- Food type: the stock fish were fed commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test

ACCLIMATION
- Acclimation period: fish were acclimatised to test conditions from 31 March 2010 to 11 April 2010

- Acclimation conditions (same as test or not): fish were maintained in a glass fibre tank with a "single pass" water renewal system. 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 14°C with a dissolved oxygen content of greater than or equal to 10.1 mg O2/L. These parameters were recorded daily

- Health during acclimation (any mortality observed): there was zero mortality in the 7 days prior to the start of the test
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure.
Hardness:
The water had a total hardness of approximately 140 mg/L (as CaCO3)
Test temperature:
Temperature maintained at approximately 14ºC.
The water temperature was recorded daily throughout the test.
pH:
pH was recorded daily throughout the test.
pH range 7.8-8.5.
Dissolved oxygen:
The dissolved oxygen concentrations were recorded daily throughout the test
The dissolved oxygen concentration was measured using a dissolved oxygen meter.
No treatment related differences for oxygen concentration were observed.
Nominal and measured concentrations:
Nominal loading rates of 10, 18, 32, 56 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 20 litre glass exposure vessels
- Type (delete if not applicable): covered
- Aeration: The test vessels were aerated via narrow bore glass tubes
- Renewal rate of test solution (frequency/flow rate): 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 material remained near nominal and to prevent the build up of nitrogenous waste products
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
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 then 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 2 (see attached background material).

OTHER TEST CONDITIONS
- Photoperiod: photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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.





Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
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 1.

There were no mortalities in the fish exposed to 10, 18, 32, 56 and 100 loading rate WAFs for a period of 96 hours. Inspection of the mortality data gave the following results:
Time (h) LL*50 (mg/L)
3 >100
6 >100
24 >100
48 >100
72 >100
96 >100

*LL = lethal loading rate.

The results of the definitive test showed the No Observed Effect Loading rate (NOEL) to be 100 mg/L loading rate WAF. The No Observed Effect Loading rate is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this loading rate.

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

- Sub-lethal effects:
There were no sub-lethal effects of exposure observed in the test.

- Physico-chemical measurements:
The results of the physico-chemical measurements are given in Appendix 4 (see attached background material). Temperature was maintained at approximately 14 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

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 the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.

- Vortex depth measurements:
The vortex depth was recorded at the start and end of each mixing period and was observed to be a dimple at the water surface on each occasion (see Table 2).

- Observations on test material solubility:
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of each mixing period the 10, 18, 32, 56 and 100 mg/L loading rates were observed to be slightly cloudy water columns. At the end of each mixing period, and after the 1-hour settlement period the 10, 18, 32, 56 and 100 mg/L loading rates were observed to be slightly cloudy water columns with some test material at the base of the vessel. Microscopic examination of the WAFs showed micro-particles to be present and therefore it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all micro-particles. During the test the 10, 18, 32, 56 and 100 mg/L loading rates were observed to be clear, colourless solutions.

- Chemical analysis of test loading rates:
Analysis of the fresh media at 0 and 72 hours (see Appendix 3 - attached background material) showed measured concentrations of manganese to range from 0.616 to 6.67 mg/L. Analysis of the old media at 24 and 96 hours showed measured concentrations of manganese to range from 0.526 to 5.21 mg/L.
The dissolved test material may have been one or several components of the test material. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole, the results were based on nominal loading rates only.





















Results with reference substance (positive control):
Not applicable.
Sublethal observations / clinical signs:

Result Tables:

Table 1: Cumulative Mortality Data in the Definitive Test

Nominal

Loading Rate

(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

10

0

0

0

0

0

0

0

18

0

0

0

0

0

0

0

32

0

0

0

0

0

0

0

56

0

0

0

0

0

0

0

100

0

0

0

0

0

0

0

Table 2:  Vortex Depth Measurements at the Start and End of Each Mixing Period

FIRST MIXING PERIOD

 

Nominal Loading Rate (mg/L)

Control

10

18

32

56

100

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.5

36.5

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

SECOND MIXING PERIOD

 

Nominal Loading Rate (mg/L)

Control

10

18

32

56

100

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

 

* Start of mixing period

+ End of mixing period

THIRD MIXING PERIOD

 

Nominal Loading Rate (mg/L)

Control

10

18

32

56

100

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

36.0

36.0

37.0

37.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

FOURTH MIXING PERIOD

 

Nominal Loading Rate (mg/L)

Control

10

18

32

56

100

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present


* Start of mixing period

+ End of mixing period

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material to the freshwater fish, rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-hour LL50 value of greater than 100 mg/L loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Based on the mean measured test concentrations as test item the acute toxicity of the test item to rainbow trout gave a 96-Hour LC50 value of greater than 1.2 mg/l. The No Observed Effect Concentration was 1.2 mg/l.

This study showed that there were no toxic effects at saturation.
Executive summary:

A study was performed to assess the acute toxicity of the test material 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.

During the study, fish were exposed, in groups of seven, to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/L for a period of 96 hours at a temperature of approximately 14°C under semi-static 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.

Based on nominal loading rates, the 96 -hour LL50 was greater than 100 mg/L loading rate WAF and correspondingly 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.

Description of key information

96-hour LL50 >100 mg/L loading rate WAF, 96-hour NOEC = 100 mg/L loading rate WAF, OECD 203, EU Method C.1, Priestly & Mullee (2010)

Key value for chemical safety assessment

Additional information

A study was performed to assess the acute toxicity of the test material 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.

During the study, fish were exposed, in groups of seven, to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/L for a period of 96 hours at a temperature of approximately 14°C under semi-static 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.

Based on nominal loading rates, the 96 -hour LL50was greater than 100 mg/L loading rate WAF and correspondingly 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.