Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

29 January 1991 to 2 February 1991

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.

Qualifier:

according to guideline

Guideline:

other: OECD Test Guidelines

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA/OTS guidelines for testing environmental effects of chemicals (ES EPA, 1985, 1987)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

No analytical sampling

Vehicle:

no

Details on test solutions:

Exposure Solution Preparation
Prior to test initiation, appropriate aliquots of the test materail were added to the mixing chamber in each test vessel to prepare the test concentrations. These solutions were stirred vigorously overnight (approximately 20 hours). During the mixing process, the water soluble fraction of the test material was slowly dispersed through the nylon screened slits of the mixing chamber and into the exposure section of the test vessel. After approximately 20 hours of stirring, the test organisms were added to the test vessels and the test was initiated. The solution contained in the mixing chamber was continuously stirred throughout this procedure and for the first 24-hour exposure period. At test initiation, a second set of exposure vessels was established in the same manner as the first set and stirring was initiated. This set of exposure vessels was used as the "new" solution at the 24-hour renewal period. Once again, a second set of exposure vessels was established at this time to be used at the next renewal period. This process was repeated at 48 and 72-hours. All test vessels were cleaned with a commercial solvent mixture, warm soapy water and rinsed with clean dilution water.

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

Test Organisms
The sheepshead minnow (SLl lot# 91A2) were obtained from the SLI Brood Unit and held in a 500-L fiberglass tank under a photoperiod of 16 hours light and 8 hours darkness. A closed loop recirculating filtration system provided. natural seawater with a salinity range of 30 - 36%, a pH range of 7.5 - 7.8 and a dissolved oxygen concentration range of 65- 89% of saturation (Weekly Record of Fish Holding Water Characteristics). The fish were maintained under these conditions for a minimum of 14 days prior to testing. The temperature in the holding tank ranged from 21 - 22 °C during this 14-day period. The fish were fed a dry commercial flake food; ad libitum, except during the 48 hours prior to testing. Routine analyses were conducted on representative samples of the food source for the presence of pesticides and PCBs (attached Appendix 11). Food sources are considered to be of acceptable quality if the total, concentration of pesticides measured is less than 0.3 mg/kg (ASTM, 1985). There was no mortality of the test fish population during the 48-hours prior to testing (Daily Record of Fish Holding Conditions). A representative sample (N =30) of fish from the test population had a mean wet weigh of 0.37 g (range 0.19 - 0.90 g) and a mean total length of 27 mm (range 21 - 39 mm) (Fish Weight and Length Log).

Dilution Water
The dilution water used during this study was natural seawater collected from, the Cape Cod Canal, Bourne, Massachusetts. Then seawater was transferred by a pump (fIberglass reinforced thermoplastic housing), through polyvinyl chloride (PVC) pipes and transported to the laboratory in a 3400-L fiberglass tank. In the laboratory the seawater was passed through a series of polypropylene core filters (20- and 5-micron) and then recirculated within an epoxy-lined concrete reservoir prior to use. The filtered seawater was pumped to the laboratory under constant pressure through PVC pipes, an activated carbon filter and a polypropylene heat exchanger system.

The dilution water quality was biologically monitored in the laboratory through the maintenance of continuous cultures of mysids (Mysidopsis bahia). Satisfactory survival, growth, and reproduction of these sensitive organisms are used to substantiate the quality and acceptability of natural seawater used for testing marine organisms at the SLl facility. In accordance with EPA-GLP, routine analyses are conducted on representative samples of the seawater , for the presence of pesticides and PCBs. None of these compounds have been detected in any of the water samples analysed, in agreement with US EPA and ASTM standard practices (attached Appendix Ill). In addition, representative samples of the dilution water source were analysed monthly for the total organic carbon (TOC) concentration. Based on these analyses, the TOC concentration of the dilution water source was 2.9 mg/L for the month of January 1991. The results of these analyses and the ability of mysids to survive and reproduce over several culture generations in this water source, confirmed that the dilution water used during this test was of acceptable quality.

Test type:

semi-static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

All test vessels were examined at 24, 48, 72 and 96 hours of exposure as follows: mortalities were recorded, dead fish were removed,and observations of the fish and the physical characteristics of the test solutions were recorded.

Hardness:

no data

Test temperature:

Test aquaria were placed in a waterbath designed to maintain test solution temperature at 23 °C. Temperature was measured in each treatment level and control solution at test initiation. Daily thereafter, these parameters were measured in both the ages (approximately 24 hours old) and freshly prepared test solutions. Daily temperature was measured with a Brooklyn alcohol thermometer, while, continuous temperature monitoring was performed using a Brooklyn Min/Max thermometer. The temperature measurements recorded during the definitive toxicity test are presented in Table 1. Daily and continuous monitoring of the test solutions determined that the test solution temperature was maintained at 23 °C during the exposure period.

pH:

pH was measured in each treatment level and control solution at test initiation. Daily thereafter, these parameters were measured in both the ages (approximately 24 hours old) and freshly prepared test solutions. Measurements of pH were made with a LaMotte Model HA pH meter and combination electrode. The pH measurements recorded during the definitive toxicity test are attached in Table 2.

Dissolved oxygen:

Dissolved Oxygen Concentration was measured in each treatment level and control solution at test initiation. Daily thereafter, these parameters were measured in both the ages (approximately 24 hours old) and freshly prepared test solutions. Dissolved oxygen concentration was measured with a YSI Model #57 dissolved oxygen meter and probe. The dissolved oxygen concentration measurements recorded during the definitive toxicity test are presented in Table 1.

Salinity:

Salinity was measured in each treatment level and control solution at test initiation. Daily thereafter, these parameters were measured in both the ages (approximately 24 hours old) and freshly prepared test solutions. Salinity concentrations presented in this report were measured with an Argent refractometer. The salinity measurements recorded during the definitive toxicity test are presented in attached Table 1. Daily salinity measurements of the test solutions ranged from 31 - 32%.

Nominal and measured concentrations:

In the Tier I test sheepshead minnow were exposed to a single concentration of 1000 mg/l.
In the preliminary test (Tier III) sheepshead minnow were exposed to nominal concentrations ranging from 1.0 to 100 mg/l.
Definitive test: 1.3, 2.2, 3.6, 6.0 and 10 mg/l

Details on test conditions:

Test System
The test solution depth was 18.4 cm with a surface area of 819 cm2. Each aquarium contained a glass cylindrical mixing chamber which served to mix and disperse the test material. Each chamber measured 4 X 11 inches and contained three 1/4 X 2 inch slits located about 1 inch from the chamber bottom. Each slit was covered with 0.363 mm Nytex screen to prevent insoluble test material from entering the exposure section of the test vessel. Each mixing chamber was secured 2 inches from the bottom of the test vessel. A Tamco model 700 laboratory mixer was used, to stir the test material in the glass mixing chamber. All test vessels were labeled to identify test material and treatment level. Test aquaria were placed in a waterbath designed to maintain test solution temperature at 23°C.

Test Initiation
The test was initiated when ten sheepshead minnow were impartially selected from the holding tank and placed in each test aquaria, two at a time until each aquaria contained 10 fish. The resulting test organism loading concentration was 0.25 grams of biomass per liter of test solution. Fish were not fed during the exposure. The test organisms, were transferred into newly prepared test solution at the 24, 48, and 72 hour observation periods. Renewals were not performed if 100% mortality was observed among fish in the treatment level solutions .

The test area was illuminated with Durotest Yitalite (R) fluorescent lights at an intensity of 28 footcandles at the solution's surface. Sudden transitions from light to dark and vice versa were avoided. The photoperiod during the test was the same as in the culture area.

Test Monitoring
All test vessels were examined at 24, 48, 72 and 96 hours of exposure as follows: mortalities were recorded, dead fish were removed,and observations of the fish and the physical characteristics of the test solutions were recorded. The test was terminated if 100% mortality was observed among fish in all treatment Ievel solutions.

Specific conductivity was measured with a yellow Springs Instrument Company (YSI) model #33 salinity-conductivity-temperature meter and probe. Light intensity was measured with a General EIectric type #214 light meter.

The nominal test concentrations and the corresponding mortality data derived from the toxicity test were used to empirically estimate the median lethal concentration (LC50) and 95% confidence interval for each 24-hour interval of the exposure period. TheLC50 is defined as the concentration of:the test material in dilution water, lethal to 50% of the test organism population at the stated time interval. If at least one test concentration caused mortality of greater than or equal to 50% of the test population, then a computer program modified from the program of C. Stephan (Peltier, 1985) was used to calculate the LC50 values arid 95% confidence intervals.

Reference substance (positive control):

not specified

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

3.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limits of 2.6 - 4.3 mg/l

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.2 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:

Tier I
A Tier I toxicity test was conducted from 10 -11 April 1990, exposing sheepshead minnow to the test material at a single concentration of 1000 mg/L. This exposure solution and a dilution water control were maintained in triplicate. The Tier I study was conducted simultaneously in the same test system used during the Tier I exposure of mysid shrimp to the test material (SLI Study# 1479.1189.6115.510). Due to complete organism mortality in all replicates of the test solution at the 24-hour interval, the toxicity test was terminated. At the request of the Study Sponsor, Tier 11 testing was omitted and Tier III testing was initiated.

Tier II
Preliminary Test.
Prior to initiating, the Tier III definitive study, a preliminary range finding test was conducted at SLI. During this range finding, test sheepshead minnow were exposed under static conditions to nominal concentrations of the test material ranging from 100 to.1.0 mg/L. Following 96 hours of exposure, 100,% mortality was observed in !he two highest treatment levels tested (100 and 10 mg/L), while mortality of 10% was observed in the 1.0 mg/L treatment level. Based on the results of the range finding test, nominal concentrations of 10, 6.0, 3.6, 2.2 and 1.3 mg/L of the test material were selected for the definitive study. The Tier III range finding test was conducted simultaneously in the same test system used during the preliminary exposure of mysid shrimp to the test material (SLI Study # 1479.1189.6115.510).

Definitive Test.
Water quality parameters remained within acceptable ranges for the survival of sheepshead minnow and were not affected by the concentrations of the test material tested.

The nominal test concentration, corresponding cumulative mortalities and the observations made during the acute exposure are presented in attached Table 3. Throughout the exposure period, no sign of undissolved material (i.e., film on the solutions surface, precipitate) was observed in any of the exposure solutions. Following 96 hours of exposure, 100% mortality was observed among fish exposed to the two highest treatment levels (10 and 6.0 mg/L). Mortality of 40% was observed among fish exposed.to the 3.6 mg/L treatment level, with sublethal (e.g. lethargy) were observed among surviving fish exposed to this treatment level. No significant (> 10%) mortality or sublethal effects were recorded in the two lowest concentrations (2.2 and 1.3 mg/L) of the test material tested. Based on this data, it was concluded that the results of this study were clearly concentration dependent. Based on these results, the 96-hour LC50 value (95% confidence interval) for sheepshead minnow exposed to the test material was calculated by probit analysis to be 3.3 (2.6 ~ 4.3) mg/L. The NOEC established for this study was 2.2 mg/L.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Three statistical methods were available in the computer program: moving average angle analysis, probit analysis, and nonlinear interpolation with 95% confidence intervals calculated by binomial probability. Moving average angle and probit analyses yield statistically sound results only it at least two concentrations produce a mortality of between 0 and 100% of the test organism population. The selection of reported LC50 values and 95% confidence intervals, was based upon an examination of the data base and the results of the computer analysis. Selection criteria included the establishment of a concentration-effect (mortality) relationship, the number of concentrations causing partial responses and the span of responses bracketing the LC50 value.

If two or more statistical methods produced acceptable results, then the method which yielded the smallest 95% confidence interval was selected. The No Observed Effect Concentration (NOEC) was also determined during the 96-hour exposure and is defined as the highest concentration tested at and below which there were no toxicant-related mortalities or physical and behavioral abnormalities (e.g. lethargy, loss of equilibrium, darkened pigmentation).

Validity criteria fulfilled:

yes

Conclusions:

The 96-hour LC50 value (95% confidence interval) for sheepshead minnow exposed to the test material was calculated by probit analysis to be 3.3 (2.6 ~ 4.3) mg/L. The NOEC established for this study was 2.2 mg/L.

Executive summary:

TEST PROTOCOL: Protocol for Conducting a Static Renewal Acute Toxicity Test with Sheepshead Minnow (Cyprinodon variegatus) Following OECD Guidelines,SLI Protocol #:081787/72.3 SM-SA and Protocol Amendment # 1 signed by the Study Director on 15 February 1991

REPORT NUMBER: 90 -12 -3585

STUDY NUMBER: 1479.1189.6116.500

DATE RECEIVED: 8 OCTOBER 1990

DESCRIPTION: A light gold liquid, tested as 100% active ingredient

TEST DATES: 29 January - 2 February 1991

SPECIES: Sheepshead minnow, Cyprinodon variegatus

Total length: Mean = 27 mm; range; = 21 - 39 mm; N = 30

Wet weight: Mean =0.37 g; range: = 0.19 - 0.90 g; N =30

Source: SLI Brood Unit

DILUTION WATER: Natural filtered seawater from Cape Cod Canal, Bourne, Massachusetts

TEST TEMPERATURE: 23 Deg C

NOMINAL TEST CONCENTRATION: 10, 6.0, 3.6, 2.2 and 1.3 mg/l

RESULTS: The 96-hour LC50 value (95%, confidence interval) was calculated by probit analysis to be 3.3 (2.6 - 4.3) mg/L. The NOEC established for this study was 2.2 mg/L

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The range-finding tests were conducted between 26 May 2002 and 6 July 2002 and the definitive test between 14 July 2002 and 19 July 2002.

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

referenced as Method C.1 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC)

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 materials, 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 material in cases where the test material 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 material with water for a prolonged period. Previous experience gained from studies conducted on poorly water soluble test materials has shown that a mixing period of 24 - 48 hours is sufficient to ensure equilibration between the test material and water phase. At the completion of mixing, the test material phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test material and/or leachates from the test material). Exposures are expressed in terms of the original concentration of test material in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test material in the WAF.

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

The concentration, homogeneity and stability of the test material in the test preparations were not determined at the request of the Sponsor.

Vehicle:

no

Details on test solutions:

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

Amounts of test material (200 and 2000 mg) were each separately added to the surface of 20 litres of dechlorinated tap water to give the 10 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 slight dimple at the water surface. These were stirred for 24 hours. The stirring was stopped after 24 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Parafilm 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 Parafilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 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 material to be present, therefore a glass wool plug was not used to filter the WAFs.

Due to 100% mortality being observed at all the loading rates employed during the test, a further range-finding test was carried out at loading rates of 1.0 and 10 mg/l in order to obtain a No-Observed Effect Loading rate.
Amounts of test material (21 and 210 mg) were each separately added to the surface of 21 litres of dechlorinated tap water to give the 1.0 and 10 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 slight dimple at the water surface. These were stirred for 24 hours. The stirring was stopped after 24 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Parafilm 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 Parafilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 1.0 and 10 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present, therefore a glass wool plug was not used to filter the WAFs.

In the range-finding tests 3 fish were added to each 20 litre test and control vessel and maintained at 14.0°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 24 and 96 hours for the initial and second range-finding test respectively. Each test was carried out under static test conditions.

The control group was maintained under identical conditions but not exposed to the test material. Data from the control group was shared with similar concurrent studies in both range-finding tests. 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.

Experimental Preparation
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 (21.0, 37.8, 67.2, 117.6 and 210 mg) were each separately added to the surface of 21 litres of dechlorinated tap water to give the 1.0, 1.8, 3.2, 5.6 and 10 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 slight dimple at the water surface. These were stirred for 24 hours. The stirring was stopped after 24 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Parafilm 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 Parafilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present, therefore a glass wool plug was not used to filter the WAFs.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Rainbow trout is a freshwater fish representative of a wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems.

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 29 May 2001. Fish were maintained in a glass fibre tank with a "single pass" water renewal system. Fish were acclimatised to test conditions from 3 July 2002 to 15 July 2002. 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 14.0°C with a dissolved oxygen content of greater than or equal to 9.2 mg O2/l. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was less than 1% mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.6 cm (sd = 0.4) and a mean weight of 1.53 g (sd = 0.25) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.77 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 100 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

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. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

Hardness:

Total hardness of approximately 100 mg/l as CaCO3.

Test temperature:

The water temperature was recorded daily throughout the test.
The results of the physico-chemical measurements are given in Appendix 2, under overall remarks, attachments section. Temperature was maintained at 14.0°C throughout the test.

pH:

The pH was 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 measured using a WTW pH 320 pH meter ar.

The results of the physico-chemical measurements are given in attached Appendix 2. There were no treatment related differences for 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 study 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.

Dissolved oxygen:

The dissolved oxygen concentrations were recorded daily throughout the test. The water temperature was controlled at 14 Deg C with a dissolved oxygen content of greater than or equal to 9.2 mg O2/1.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 dissolved oxygen concentration was measured using a YSI 550 dissolved oxygen meter.

Salinity:

No Data

Nominal and measured concentrations:

Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6 and 10 mg/l.

Details on test conditions:

Exposure conditions
As in the range-finding tests 20 litre glass exposure vessels were used for each test concentration. At the start of the test 10 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 14.0°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 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 material.

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 test concentrations of soluble components of the test material 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.

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

Reference substance (positive control):

not specified

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

2.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limits of 1.8 -3.2 mg/l loading rate WAF

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

1 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:

Range-finding Tests
Cumulative mortality data from the exposure of rainbow trout to the test material during the range-finding tests are given in Tables 1 and 2 and sub-lethal effects of exposure for the second range-finding test are given in Table 3, under results incl. tables section.
The results of the initial range-finding test showed 100% mortality at the 10 and 100 mg/l loading rate WAFs. In the second range-finding test the results showed no mortalities at the 1.0 mg/l loading rate WAF. However, mortalities were observed at the 10 mg/l loading rate WAF.
Based on this information loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/l using a stirring period of 24 hours followed by a 1-Hour standing period were selected for the definitive test.

Mortality data
Cumulative mortality data from the exposure of rainbow trout to the test material during the definitive test are given in Table 4 and the relationship between percentage mortality and loading rate at 96 hours is attached in Figure 1.
An estimate of the 3–Hour LL50* values was given by inspection of the mortality data and analysis of the mortality data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 hours and the geometric mean method at 6, 24, 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 > 10 –
6 4.2 3.2 – 5.6 **
24 4.2 3.2 – 5.6 **
48 4.0 3.6 – 4.5
72 2.4 1.8 – 3.2**
96 2.4 1.8 – 3.2**

* LL = Lethal Loading rate
** Loading rates resulting in 0% and 100% mortalities respectively

The results of the definitive test showed the highest loading rate resulting in 0% mortality to be 1.8 mg/l loading rate WAF, the lowest loading rate resulting in 100% mortality to be 3.2 mg/l loading rate WAF and the No Observed Effect Loading rate (NOEL) to be 1.0 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 relationship between the median lethal loading rate (LL50*) and time is presented graphically in Figure 2, attached.

Sub-lethal effects
Sub-lethal effects of exposure were observed at the 1.8 mg/l loading rate WAF and above. These responses were swimming at the bottom, swimming at the surface, increased pigmentation, increased pigmentation with swimming at the bottom, increased pigmentation with loss of equilibrium and the presence of moribund fish (see Table 5, under results incl. tables section).
After approximately 3 hours exposure all fish at the 5.6 and 10 mg/l test concentrations were observed to be moribund. Due to the approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) these fish were killed and were classed as mortalities for the 6 Hour time point.
After approximately 45.5 and 69.5 hours exposure 1/10 and 9/9 fish respectively in the 3.2 mg/l loading rate were observed to be moribund. Due to the approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) these fish were killed and were classed as mortalities for the 48 and 72 Hour time points respectively.

Vortex depth measurements
The vortex depth was recorded at the start and end of each mixing period and was observed to be a slight dimple at the water surface on each occasion (see Table 6, under results incl. tables 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 of the mixing all the loading rates were observed to be clear colourless water columns with clear colourless oily globules at the water surface. At the end of each mixing period and after the 1-Hour settlement period all the loading rates were observed to be clear colourless water columns with clear colourless oily globules at the water surface and on the bottom of the vessel. After siphoning and for the duration of the test, all the loading rates were observed to be clear, colourless solutions. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present, therefore a glass wool plug was not used to filter the WAFs.

Results with reference substance (positive control):

- Results with reference substance valid?
Not applicable

- Mortality:
Not applicable

- LC50:
Not applicable

- Other:
Not applicable

Reported statistics and error estimates:

An estimate of the LL50*values was given by inspection of the mortality data at 3 and 6 hours.
The LL50*values and associated confidence limits at 72 and 96 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al1977) using the ToxCalc computer software package (ToxCalc 1999) and at 24 and 48 hours the LL50* values were calculated using the geometric mean method as follows:

*LL = Lethal Loading rate

LL50 * value = √L1 x L2


Where:
L1= loading rate showing 0% mortality L2= loading rate 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 loading rate showing no lethality and the lowest loading rate showing 100% lethality is calculated. The loading rates resulting in 0% and 100% mortality will be the 95% confidence limits.
Analysis of the mortality data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 72 and 96 hours and the geometric mean method.

Sublethal observations / clinical signs:

Table1: Cumulative Mortality Data in the Initial Range-finding Test

Nominal Loading Rate (mg/l)	Cumulative Mortality (Initial Population = 3)
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours
Control	0	0	0	-	-	-
10	3*	3	3	-	-	-
100	3	3	3	-	-	-

- Test terminated due to 100% mortality*After approximately 1 hour exposure all 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 3-Hour time point.

Table 2: Cumulative Mortality Data in the Second Range-finding Test

Nominal Loading Rate (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
1.0	0	0	0	0	0	0
10	0	3*	3	3	3	3

*After approximately 3 hours exposure all fish were observed to be moribund with arched spines. Due to the approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities at the 6-Hour time point

Table 3: Sub-lethal Effects of Exposure in the Second Range-finding 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
10	Moribund with arched spines	3/3*	A/D

*After approximately 3 hours exposure all fish were observed to be moribund with arched spines. Due to the approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities at the 6-Hour time pointA/D= All fish dead

Table 4: Cumulative Mortality Data in the DefinitiveTest

Nominal Loading Rate (mg/l)	Cumulative Mortality (Initial Population =10)						% 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
1.8	0	0	0	0	0	0	0
3.2	0	0	0	1**	10***	10	100
5.6	0	10*	10	10	10	10	100
10	0	10*	10	10	10	10	100

  * After approximately 3 hours all the 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 6-Hour time point

**After approximately 45.5 hours exposure 1/10 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 48-Hour time point

***After approximately 69.5 hours exposure 9/9 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 72-Hour time

point.

Table 5: 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
1.0	No abnormalities detected
1.8	Swimming at bottom Increased pigmentation and swimming at bottom Swimming at surface						3/10 1/10   6/10
3.2	Increased pigmentation with swimming at bottom Increased pigmentation with loss of equilibrium Increased pigmentation			1/10   1/10	7/9**       2/9	A/D***
5.6	Moribund	10/10	A/D*
10	Moribund	10/10	A/D*

*After approximately 3 hours all the 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 6-Hour time point

**After approximately 45.5 hours exposure 1/10 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 48-Hour time point

***After approximately 69.5 hours exposure 9/9 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 72-Hour time point

A/D= All fish dead

 

Table 6: Vortex Depth Measurements at the Start and End of Each Mixing Period

FIRST MIXING PERIOD

Nominal Loading Rate (mg/l)

Control

1.0

1.8

3.2

5.6

10

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

36

36

36

36

36

36

36

36

36

36

36

36

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

-Not used due to 100% mortalities at this loading rate

SECOND MIXING PERIOD

Nominal Loading Rate (mg/l)

Control

1.0

1.8

3.2

5.6

10

*

+

*

+

*

+

*

+

*

+

*

+

Height of Water Column (cm)

36

36

36

36

36

36

36

36

36

-

36

-

Depth of Vortex (cm)

~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

-

THIRD MIXING PERIOD

	Nominal Loading Rate (mg/l)
	Control		1.0		1.8		3.2
	*	+	*	+	*	+	*	+
Height of Water Column (cm)	36	36	36	36	36	36	36	36
Depth of Vortex (cm)	~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

FOURTH MIXING PERIOD

	Nominal Loading Rate (mg/l)
	Control		1.0		1.8		3.2
	*	+	*	+	*	+	*	+
Height of Water Column (cm)	36	36	36	36	36	36	36	-
Depth of Vortex (cm)	~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	-

*= Start of mixing period

+= End of mixing period

-= Not used due to 100% mortalities at this loading rate

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 2.4 mg/l loading rate WAF with 95% confidence limits of 1.8 – 3.2 mg/l loading rate WAF. The No Observed Effect Loading rate was 1.0 mg/l loading rate WAF.
* LL = Lethal Loading rate

Executive summary:

Introduction

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 Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods

Following preliminary range-finding tests, fish were exposed, in groups of ten, to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/l  for a period of 96 hours at a temperature of 14.0ºC 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 & Conclusion

The 96-Hour LL₅₀*based on nominal loading rates was 2.4 mg/l loading rate WAF with 95% confidence limits of 1.8 – 3.2 mg/l loading rate WAF. The No Observed Effect Loading rate was 1.0 mg/l loading rate WAF.

*LL = Lethal Loading rate

Description of key information

LL50 = 2.4 mg/L, NOEL = 1 mg/L; study performed in line with OECD Guideline 203; Sewell (2002)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2.4 mg/L

Marine water fish

Marine water fish

Effect concentration:

3.3 mg/L

Additional information

GLP studies on short-term toxicity to fish are available for rainbow trout and sheepshead minnow. Both studies are performed to GLP and are stated to be in line with standardised guidelines. The study using rainbow trout is considered to be the key study on the basis that it is performed using freshwater and the results values obtained from this study are more 'worst-case'.

In the key study, rainbow trout fish were exposed, in groups of ten, to Water Accommodated Fractions (WAFs) of the test substance over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L for a period of 96 hours at a temperature of 14.0ºC 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. The 96-Hour LL₅₀ based on nominal loading rates was 2.4 mg/L loading rate WAF with 95% confidence limits of 1.8–3.2 mg/L loading rate WAF. The No Observed Effect Loading rate was (NOEL) 1 mg/L loading rate WAF.

In the supporting study, sheepshead minnow were exposed, in groups of ten, to Water Accommodated Fractions (WAFs) of the test substance over a range of nominal loading rates of 1.3, 2.2, 3.6, 6.0 and 10 mg/L for a period of 96 hours at a temperature of 23.0ºC under semi-static conditions in a definitive test. Mortalities were recorded, dead fish were removed and observations of the fish were made at 24, 48, 72 and 96 hours after exposure. The 96 hour LC50 was 3.3 mg/L with 95% confidence limits of 2.6-4.3 mg/L. The NOEC was 2.2 mg/L