Reaction Products of C4 alcohols and C4 alkenes obtained as by-products from the manufacturing of butan-2-ol by sulfuric acid esterification and hydrolysis of butene

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 January 2010 and 23 February 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

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Products of C4 alcohols and C4 alkenes obtained as by-products from the manufacturing of butan-2-ol by sulfuric acid esterification and hydrolysis of butene

EC Number:

701-242-6

Molecular formula:

A complex and variable combination of C4 hydrocarbons, 2,2'-oxybisbutane and butan-2-ol.

IUPAC Name:

Reaction Products of C4 alcohols and C4 alkenes obtained as by-products from the manufacturing of butan-2-ol by sulfuric acid esterification and hydrolysis of butene

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, 48, 72 (old and fresh media) and 96 hours (old media) for quantitative analysis. Duplicate samples were taken and stored at approximately -20°C for further analysis, if necessary. The method of analysis, stability, recovery and test solution analyses are described in attached Appendix 3.

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 definitive test the test item was prepared as a Water Accommodated Fraction (WAF).
The loading rates to be used in the definitive test were determined by a preliminary range-finding test. In the range-finding test fish were exposed to a series of nominal loading rates of 10 and 100 mg/l. The test item was prepared as a WAF.
Amounts of test item (210 and 2100 mg) were each separately added to the surface of 21 litres of dechlorinated tap water to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the preparations were stirred for 23 hours. 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 theNescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning to give the 10 and 100 mg/l loading rate WAFs.
In the range-finding test three fish were added to each 20 litre test and control vessel and maintained at approximately 14°C 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.
Due to the possible volatile nature of the test item the test was conducted using completely filled and sealed vessels. The test preparations were transferred to these vessels after approximately 2 hours exposure as the media was initially placed in vessels which were covered only. Given the short period unsealed, this was considered not to affect the test. 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.

Definitive test
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.

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. These were stirred for 23 hours in sealed vessels due to the volatile nature of the test item. 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 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.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media) (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 and dissolved oxygen concentration were 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 each 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 6 January 2010. Fish were maintained in a glass fibre tank with a "single pass" water renewal system. Fish were acclimatised to test conditions from 3 February 2010 to 15 February 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 14°C with a dissolved oxygen content of greater than or equal to 10.2 mg O2/l. These parameters were recorded daily, with the exception of a single oxygen reading which was missed in error. The stock fish were fed commercial trout pellets which was discontinued approximately 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 5.0 cm (sd = 0.6) and a mean weight of 1.85 g (sd = 0.55) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.65 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 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 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 was measured using a WTW pH/Oxi 340I pH.
pH range of 7.8-8.0.
Please see Physico-Chemical Measurements appendix 4 in any other information on materials and methods section.

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 methods section
for dissolved oxygen results.

Salinity:

Freshwater used.

Nominal and measured concentrations:

The loading rates to be used in the definitive test were determined by a preliminary range-finding test. In the range-finding test fish were exposed to a series of nominal loading rates of 10 and 100 mg/l. The test item was prepared as a WAF. 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.

Details on test conditions:

TEST SYSTEM
- Test vessel:
As in the range-finding test 20 litre glass exposure vessels were used for each test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. Due to the volatile nature of the test item the test was conducted using completely filled and sealed round bottomed glass exposure vessels. The vessels were maintained at approximately 14¿C 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 not 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 test concentrations of soluble components of the test item were maintained over the test 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 2 in any other information on materials and method section.

- 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 concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

61 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limits of 43 - 97 mg/l loading rate WAF

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: CL not stated

Details on results:

Validation of Mixing Period
Pre-study investigational 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 by extending the preparation period for longer than 24 hours. Therefore for the purpose of testing, the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour standing period.

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 and sub-lethal effects of exposure are given in Table 2 see in any other information on results section.
The results showed no mortalities at the 10 and 100 mg/l loading rate WAFs. However, sub-lethal effects of exposure were observed at the 100 mg/l loading rate WAF. Increased pigmentation was observed at a loading rate of 10 mg/l after 24 hours exposure. However, this effect was not observed again throughout the duration of the test indicating that the effect may not be due to a toxic effect.
Based on this information loading rates of 10, 18, 32, 56 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 3 see in any other infor mation on results section and the relationship between percentage mortality and loading rate at 96 hours is given in Figure 1 see in attached section.
Inspection of the mortality data at 3, 6 and 24 hours and analysis of the mortality data by the probit method (Finney 1971) at 48, 72 and 96 hours based on the nominal loading rates gave the following results:


Time (h) LL50*(mg/l) 95% Confidence Limits
(mg/l)
3 >100 -
6 >100 -
24 >100** -
48 76 55 - 140
72 71 50 - 120
96 61 43 - 97
The results of the definitive test showed the highest loading rate resulting in 0% mortality to be 32 mg/l loading rate WAF, the lowest loading rate resulting in 100% mortality to be greater than 100 mg/l loading rate WAF and the No Observed Effect Loading rate (NOEL) to be 18 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.
The results of the definitive test differed slightly from the range-finding test in that mortalities were observed at 100 mg/l loading rate WAF. This was considered to be due to the use of a semi-static test regime in the definitive test which maintained a higher level of the test item throughout the duration of the test.
The relationship between the median lethal loading rate (LL50*) and time is presented graphically in Figure 2 see in attached section.

Sub-lethal effects
Sub-lethal effects of exposure were observed at the 32 mg/l loading rate WAF and above. These responses were increased pigmentation, swimming at the surface, loss of equilibrium, lethargy and the presence of moribund fish (see Table 4 in any other information on results section.
After 6 hours exposure two out of seven fish at 100 mg/l loading rate WAF were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following observational time point.
After 24 hours exposure two out of four fish at 100 mg/l loading rate WAF and two out of seven fish at 56 mg/l loading rate WAF were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following observational time point.

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.
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 in the control fish.
The oxygen concentration in some of the test vessels was observed to be below 60% ASV in the old media. This was considered to be due to the use of completely filled and sealed test vessels which received no aeration for the duration of the test due to the suspected volatile nature of the test item. As no adverse effects were observed in the control fish throughout the duration of the test this deviation was considered not to have affected the outcome of the test.

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 5 in any other information on results section).

Observations on test material solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start and 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 clear, colourless water columns with oily globules of test item floating at the surface. After siphoning and for the duration of the test, the 10, 18, 32, 56 and 100 mg/l loading rates were observed to be clear, colourless solutions. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

Chemical analysis of test loading rates
Analysis of the freshly prepared test media at 0, 24, 48 and 72 hours (see Appendix 3 in attached section) showed measured concentrations to range from 8.13 to 94.9 mg/l. Analysis of the old media at 24, 48, 72 and 96 hours showed measured concentrations of 8.29 to 85.8 mg/l.
The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or 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):

- Results with reference substance
Not applicable

Reported statistics and error estimates:

An estimate of the LL50* values at 3, 6 and 24 hours was given by inspection of the mortality data.
The LL50* values and associated confidence limits at 48, 72 and 96 hours were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table1              Cumulative Mortality Data in the Range-finding Test

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	6	24	48	72	96
Control	No abnormalities detected
10	Increased pigmentation			3/3
100	Increased pigmentation and swimming at the surface	3/3	3/3	3/3	3/3	3/3	3/3

Table3              Cumulative Mortality Data in the DefinitiveTest

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**	2	3	5	71
100	0	0[1]	3***	5	5	5	71

[1]After 6 hours exposure two out of seven fish were observed to be moribund. 

** After 24 hours exposure two out of seven fish were observed to be moribund. 

*** After 24 hours exposure two out of four 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 for the following time point.

Table 5              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)

40.0

40.0

40.0

40.0

40.0

40.0

40.0

40.0

40.0

40.0

40.0

40.0

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

10

18

32

56

100

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

40.0

40.0

40.0

40.0

40.0

40.0

40.0

40.0

40.0

40.0

40.0

40.0

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

Table 5 (continued)          Vortex Depth Measurements at the Start and End of Each Mixing Period

THIRD MIXING PERIOD

Nominal Loading Rate (mg/l)

Control

10

18

32

56

100

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

40.0

40.0

40.0

40.0

40.5

40.5

40.0

40.0

40.0

40.0

40.0

40.0

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)

40.5

40.5

40.0

40.0

40.0

40.0

40.0

40.0

40.5

40.5

40.0

40.0

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

Table4              Sub-lethal Effects of Exposure in the DefinitiveTest

Nominal Loading Rate (mg/l)	Sub-lethal Effects	Time (Hours)
		3	6	24	48	72	96
Control	No abnormalities detected
10	No abnormalities detected
18	No abnormalities detected
32	Increased pigmentation Increased pigmentation and lethargic Increased pigmentation and swimming at the surface	7/7	7/7	7/7	7/7	6/7 1/7	6/7       1/7
56	Increased pigmentation Increased pigmentation and loss of equilibrium Moribund Increased pigmentation and lethargic Increased pigmentation and swimming at the surface	7/7	7/7	3/7 2/7   2/7**	3/5 1/5     1/5	3/4           1/4	2/2
100	Increased pigmentation Increased pigmentation and swimming at the surface Increased pigmentation and loss of equilibrium Moribund Increased pigmentation and lethargic	4/7 2/7     1/7	2/7 2/7     1/7   2/7[*]	2/4   2/4***	1/2             1/2	1/2             1/2	1/2             1/2

[*]After 6 hours exposure two out of seven fish were observed to be moribund. 

** After 24 hours exposure two out of seven fish were observed to be moribund. 

*** After 24 hours exposure two out of four 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 for the following time point.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

This study is classified as acceptable and satisfies the guideline requirements for this study end point.

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 61 mg/l loading rate WAF with 95% confidence limits of 43 - 97 mg/l loading rate WAF. The No Observed Effect Loading rate was 18 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 10, 18, 32, 56 and 100 mg/l for a period of 96 hours at a temperature of approximately 14°C in sealed vessels 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 LL₅₀*based on nominal loading rates was 61 mg/l loading rate WAF with 95% confidence limits of 43 - 97 mg/l loading rate WAF. The No Observed Effect Loading rate was 18 mg/l loading rate WAF.

Analysis of the freshly prepared test media at 0, 24, 48 and 72 hours showed measured concentrations to range from 8.13 to 94.9 mg/l. Analysis of the old media at 24, 48, 72 and 96 hours showed measured concentrations of 8.29 to 85.8 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.

*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 LL₅₀*value of 61 mg/l loading rate WAF with 95% confidence limits of 43 - 97 mg/l loading rate WAF. The No Observed Effect Loading rate was 18 mg/l loading rate WAF.

*LL = Lethal Loading rate