Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 13 January 2010 and 28 May 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
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
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, OECD 2000 and Singer et al 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. Pre-study work showed that a preparation period of 24 hours was 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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Chemical analysis of test loading rates
Water samples were taken from the control and all surviving test groups at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 (old media) hours for quantitative analysis.
Duplicate samples and samples at 24 (fresh media), 48 (fresh and old media) and 72 hours (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 3 see in attached section.

Test solutions

Vehicle:
no
Details on test solutions:
Range-finding test
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the range-finding test the test item was prepared as a Water Accommodated Fraction (WAF).
The test concentrations to be used in the initial experiment were determined by a preliminary range-finding test.
In the range-finding test fish were exposed initially to a single nominal loading rate of 100 mg/l loading rate WAF. However, shortly after exposure fish were showing signs of sub-lethal effects, therefore additional fish were exposed to a further concentration of 10 mg/l loading rate WAF. Following a review of the solubility data it was also noticed that the test item should have been treated as volatile, therefore the fish were transferred to completely filled and sealed vessels. Although some losses may have occurred during the initial period that fish were in open vessels it was considered justifiable to take this action on ethical grounds.
Amounts of test item (210 and 2100 mg) were each separately added to the surface of 21 litres of reconstituted water to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, 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 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 or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
In the range-finding test 3 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 completely filled and sealed. After 3, 5, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.

Initial Experiment
Based on the results of the range-finding test the following test loading rates were assigned to the initial experiment: 10, 18, 32, 56 and 100 mg/l.

Experimental Preparation
Amounts of test item (225, 405, 720, 1260 and 2250 mg) were each separately added to the surface of 22.5 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 item, 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 vessels were completely filled and sealed with rubber bungs and laboratory film. 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 or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 10, 18, 32, 56 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

Definitive test
Based on the results of the initial experiment the following test loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/l.

Experimental Preparation
Amounts of test item (22.5, 72, 225, 720 and 2250 mg) were each separately added to the surface of 22.5 litres of dechlorinated tap water to give the 1.0, 3.2, 10, 32 and 100 mg/l loading rates respectively. After the addition of the test item, 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 vessels were completely filled and sealed with rubber bungs and laboratory film. 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 or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 (old media) hours (see Appendix 3 in attached section).

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 a Hanna Instruments HI 93510 digital thermometer.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period.

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Test Species
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 12 May 2010 to 24 May 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 10.2 mg O2/l. These parameters were recorded daily. The stock fish were fed commercial trout pellets which were discontinued 24 hours prior to the start of the definitive test. There was zero mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.3 cm (sd = 0.1) and a mean weight of 0.90 g (sd = 0.08) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.32 g bodyweight/litre.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

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.
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 in attached section.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h

Test conditions

Hardness:

Total hardness of approximately 140 mg/l as CaCO3.
Test temperature:

The test vessels were sealed and maintained at approximately 14ºC
pH:
The pH of the control group was observed to vary between 7.7 and 8.2. 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.
The pH was measured using a WTW pH/Oxi 340I pH.

Dissolved oxygen:
The dissolved oxygen concentration was measured using a dissolved oxygen meter.
Please see Physico-Chemical Measurements appendix 4 see in any other information on materials and method section for oxygen results.
Salinity:
Freshwater used.
Nominal and measured concentrations:
Based on the results of the range-finding test the following test loading rates were assigned to the initial experiment: 10, 18, 32, 56 and 100 mg/l.
Details on test conditions:
TEST SYSTEM
- Test vessel:
As in the range-finding test 20 litre completely filled and sealed round bottomed 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 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 received no auxiliary aeration; the diluent supply only was aerated. 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.
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.

- Type of flow-through (e.g. peristaltic or proportional diluter):
Not applicable

- Renewal rate of test solution (frequency/flow rate):
Daily

- No. of organisms per vessel:
At the start of the test 7 fish were placed in each test vessel at random, in the test preparations.

-No. of vessels per concentration (replicates):
1

- No. of vessels per control (replicates):
1

- No. of vessels per vehicle control (replicates):
Not applicable

- Biomass loading rate:
Not applicable


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
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.

-Total Organic Carbon Analysis:
Please see Appendix 3 (attached background material).

- Particulate matter:
Not measured

- Metals: Not Stated
- Pesticides: Not Stated
- Chlorine: Not Stated
- Alkalinity: Not Stated
- Ca/mg ratio: Not Stated
- Conductivity: Not Stated
- Culture medium different from test medium: Not Stated
- Intervals of water quality measurement: Not Stated


OTHER TEST CONDITIONS
The test vessels were then sealed 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.


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.


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: In the range-finding test fish were exposed to a series of nominal loading rates of 10 and 100 mg/l.
- Results used to determine the conditions for the definitive study: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.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence limits of 10-32mg/l
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
3.2 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:
Validation of Mixing Period
Pre-study work (see Appendix 2 in any other information on materials and method section) indicated that there was no significant increase in the amount of total organic carbon present in the WAF by extending the preparation period for longer than 24 hours. As such, for the purposes of testing, the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour standing period prior to the removal of the aqueous phase by mid-depth siphon.

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 on result section and sub-lethal effects of exposure are given in Table 2 see in any other information on result section.
The results showed no mortalities at the test concentrations of 10 and 100 mg/l loading rate WAF. However, sub-lethal effects were observed at 100 mg/l.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/l loading rate WAF were selected for the initial experiment.

Initial Experiment
Cumulative mortality data from the exposure of rainbow trout to the test item during the initial experiment are given in Table 3 see in any other information on result section and sub-lethal effects of exposure are given in Table 4 see in any other information on result section.
The results showed mortalities at the test loading rates of 18, 32, 56 and 100 mg/l loading rate WAF. However, sub-lethal effects were observed at the lowest concentration of 10 mg/l loading rate WAF. This was significantly different from the results of the range-finding test where no effects were observed at 10 mg/l loading rate WAF. However this was considered to be possibly due to losses that occurred when the range-finding test was initially set up incorrectly.
Based on this information loading rates of 1.0, 3.2, 10, 32 and 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.

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 5 see in any other informatio on result section and the relationship between percentage mortality and concentration at 96 hours is given in Figure 1 please see in attached section.
Analysis of the mortality data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 and 72 hours and the geometric mean method at 96 hours based on the nominal test concentrations gave the following results:
Time (h) LL*50 (mg/l Loading Rates WAF) 95% Confidence limits(mg/l Loading Rate WAF)
3 > 100 -
6 > 100 -
24 > 100 -
48 41 27 - 60
72 21 16 - 29
96 18 10 - 32**
The results of the definitive test showed the highest loading rate resulting in 0% mortality to be 10 mg/l loading rate WAF, the lowest loading rate resulting in 100% mortality to be 32 mg/l loading rate WAF and the No Observed Effect Loading rate (NOEL) to be 3.2 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 concentration (Section 5.4.2).
The relationship between the median lethal concentration (LL*50) and time is presented graphically in Figure 2 please see in attached section.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.
* LL = Lethal Loading rate
** Concentrations resulting in 0% and 100% mortalities respectively


Sub-lethal effects
Sub-lethal effects of exposure were observed at test loading rates of 10 mg/l loading rate WAF and above. These responses were increased pigmentation (see Table 6 in any other information on result section.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion (see Table 7 in any other information on result section).

Observations on test item solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of the mixing period all loading rates were observed to be clear, colourless water columns with oily globules of test item floating at the surface. After 23 hours stirring and a 1-Hour standing period the loading rates were observed to be clear, colourless water columns with an oily slick of test item at the surface. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, all the loading rates were observed to be clear, colourless solutions.

Physico-chemical measurements
The results of the physico-chemical measurements are given in Appendix 4 see in any other information on 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.

Chemical analysis of test loading rates
Analysis of the test preparations at 0 and 72 hours (fresh media) (see Appendix 3 please see in attached section) showed a concentration dependant increase in measured test concentrations ranging from less than the limit of quantitation of the analytical method, which was determined to be 0.20 mg/l, to 35.7 mg/l.
A slight decline in measured test concentrations was observed at 24 and 96 hours (old media) (see Appendix 3 please attached section) in the range of than the limit of quantitation of the analytical method to 32.0 mg/l.
Given that the 24-Hour results ranged from 75% to 97% of the 0-Hour measured test concentrations and the 96-Hour results ranged from 71% to 85% of the 72-Hour results it was considered that the decline observed in the vessels was due mainly to losses through volatility.
However, 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.
Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:
An estimate of the LL*50 values was given by inspection of the mortality data at 3, 6 and 24 hours.
The LL*50 values and associated confidence limits at 48 and 72 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999) and at 96 hours were calculated using the geometric mean method as follows:

LC50 value = vC1 x C2


where:
C1 = concentration showing 0% mortality
C2 = concentration showing 100% mortality
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.
If there are no test concentrations showing mortalities between 0% and 100%, then the geometric mean of the highest test concentration showing no lethality and the lowest test concentration showing 100% lethality is calculated. The concentrations resulting in 0% and 100% mortality will be the 95% confidence limits.

*LL = Lethal Loading rate

Any other information on results incl. tables

Table1              Cumulative Mortality Data in the Range-findingTest

Nominal

Loading Rate

(mg/l)

Cumulative Mortality

(Initial Population = 3)

3 Hours

5 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              Sub-lethal Effects of Exposure in the Range-finding Test

Nominal

Loading Rate

(mg/l)

Sub-lethal Effects

Time (Hours)

3

5

24

48

72

96

Control

No abnormalities detected

 

 

 

 

 

 

10

No abnormalities detected

 

 

 

 

 

 

100

Increased pigmentation

3/3

3/3

3/3

3/3

3/3

3/3

Table3              Cumulative Mortality Data in the InitialExperiment

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

4

57

32

0

0

0

0

0

1

14

56

0

0

0

0

2**

2

28

100

0

0

0

7[1]

7

7

100


[1]After approximately 47 hours exposure 2 fish were observed to be moribund. Due to the approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities at the 48-Hour time point.

** After approximately 71 hours exposure 1 fish was observed to be moribund. Due to the approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) this fish was killed and classed as a mortality at the 72-Hour time point.

Table4              Sub-lethal Effects of Exposure in theInitial Experiment

Nominal Loading Rate

(mg/l)

Sub-lethal Effects

Time (Hours)

3

6

24

48

72

96

Control

No abnormalities detected

 

 

 

 

 

 

10

Increased pigmentation

 

 

 

 

 

7/7

18

Increased pigmentation

Increased pigmentation and loss of equilibrium

 

 

 

 

 

7/7

 

 

7/7

 

 

 

 

3/3

32

Increased pigmentation

 

 

7/7

7/7

7/7

6/6

56

Swimming at the surface

Increased pigmentation and swimming at surface

Increased pigmentation

Increased pigmentation and loss of equilibrium

2/7

 

 

 

 

 

3/7

 

 

 

 

 

 

 

7/7

 

 

 

 

 

 

7/7**

 

 

 

 

 

1/5

 

4/5

 

 

2/5

3/5

 

 

100

Swimming at the surface

Increased pigmentation and swimming at surface

3/7

 

 

4/7

 

 

 

 

7/7*

A/D

 

 


A/D= All fish dead

*After approximately 47 hours exposure 2 fish were observed to be moribund. Due to the approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities at the 48-Hour time point.

** After approximately 71 hours exposure 1 fish was observed to be moribund. Due to the approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) this fish was killed and classed as a mortality at the 72-Hour time point.

Table5              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

1.0

0

0

0

0

0

0

0

3.2

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

32

0

0

0

2

6

7

100

100

0

0

0

7

7

7

100



Table 6              Sub-lethal Effects of Exposure in the Definitive Test

Nominal Loading Rate

(mg/l)

Sub-lethal Effects

Time (Hours)

3

6

24

48

72

96

Control

No abnormalities detected

 

 

 

 

 

 

1.0

No abnormalities detected

 

 

 

 

 

 

3.2

No abnormalities detected

 

 

 

 

 

 

10

Increased pigmentation

 

 

 

 

7/7

7/7

32

Increased pigmentation

7/7

7/7

7/7

5/5

1/1

A/D

100

Increased pigmentation

7/7

7/7

7/7

A/D

 

 

A/D= All fish dead Table 7              Vortex Depth Measurements at the Start and End of Each Mixing Period

FIRST MIXING PERIOD

 

Nominal Loading Rate (mg/l)

Control

1.0

3.2

10

32

100

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

42.0

42.0

41.5

41.5

41.5

41.5

42.0

42.0

42.0

42.0

41.0

41.0

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

SECOND MIXING PERIOD

 

Nominal Loading Rate (mg/l)

Control

1.0

3.2

10

32

100

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

42.0

42.0

41.5

41.5

41.5

41.5

42.0

42.0

42.0

42.0

41.0

41.0

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

A/D


Table 7 (continued)          Vortex Depth Measurements at the Start and End of Each Mixing Period

THIRD MIXING PERIOD

 

Nominal Loading Rate (mg/l)

Control

1.0

3.2

10

32

100

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

42.0

42.0

41.5

41.5

41.5

41.5

42.0

42.0

42.0

42.0

41.0

41.0

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

1.0

3.2

10

32

100

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

42.0

42.0

41.5

41.5

41.5

41.5

42.0

42.0

42.0

42.0

 

 

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

~0.2

A/D

A/D

Observation of Vortex

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

A/D= All fish dead

*= Start of mixing period

+= End of mixing period

A/D= All fish dead

LL = Lethal Loading rate


Applicant's summary and conclusion

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 LL 50 value of 18 mg/l loading rate WAF with 95% confidence limits of 10 - 32 mg/l loading rate WAF. The No Observed Effect Concentration was 3.2 mg/l loading rate WAF.
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 groups of seven, to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/l for a period of 96 hours at a temperature of approximately 14°C 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 LL*50 based on nominal loading rates was 18 mg/l loading rate WAF with 95% confidence limits of 10 - 32 mg/l loading rate WAF. The No Observed Effect Loading rate was 3.2 mg/l loading rate WAF.

Analysis of the test preparations at 0 and 72 hours (fresh media) showed a concentration dependant increase in measured test concentrations ranging from less than the limit of quantitation of the analytical method to 35.7 mg/l.

A slight decline in measured test concentrations was observed at 24 and 96 hours (old media) in the range of less than the limit of quantitation of the analytical method, which was determined to be 0.20 mg/l, to 32.0 mg/l.

 

Given that the 24-Hour results ranged from 75% to 97% of the 0-Hour measured test concentrations and the 96-Hour results ranged from 71% to 85% of the 72-Hour results it was considered that the decline observed in the vessels was due mainly to losses through volatility. 

However, 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.


*LL = Lethal Loading rate

Conclusion.

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LL*50 value of 18 mg/l loading rate WAF with 95% confidence limits of 10 - 32 mg/l loading rate WAF. The No Observed Effect Concentration was 3.2 mg/l loading rate WAF.


* LL = Lethal Loading rate