Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5-yl isobutyrate and 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-6-yl isobutyrate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 November 2012 - XX XXX 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study is conducted according to OECD TG 210 in compliance with GLP, without deviations that influence the quality of the results.

Justification for type of information:

This information is presented to be able to derive the acute / chronic ratio from Cyclaprop to be able to apply this to Cyclabute (see for further details the Aquatic toxicity Endpoint summary).

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Water samples were collected from one test chamber of each treatment and control group 2, 4, 9, 10, 14 and 16 days prior to test initiation to confirm the operation of the diluter. Water samples were collected from alternating replicate test chambers of each treatment and control group on Days 0, 6, 13, 20, 27 and 33 (test termination) to determine concentrations of the test substance in the test chambers. Additional samples were collected on Day 33 and stored for possible analysis if needed.
- Sampling method: All samples were collected at mid-depth in the test chambers, placed in glass vials and processed immediately for analysis.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Stock solutions were prepared four times during the test. At each preparation, a primary stock solution was prepared in HPLC-grade DMF at a nominal concentration of 25 or 50 mg/mL. Proportional dilutions of the primary stock were made in DMF to prepare additional stock solutions at nominal concentrations of 3.1, 6.3 and 13 mg/mL or 3.1, 6.3, 13 and 25 mg/mL. The stock solutions were mixed by inversion and appeared clear and colorless. Stock solutions were stored under refrigerated conditions and fresh aliquots were placed in the syringe pumps every two to four days during the test. The stock solutions were delivered to the diluter mixing chambers (at a rate of 10.0 µL/minute) where they were mixed with dilution water (at a rate of 100 mL/minute) to achieve the desired test concentrations of 0.31, 0.63, 1.3, 2.5 and 5.0 mg/L. The solvent control was prepared by injecting HPLC-grade DMF into the mixing chamber for the solvent control.
- Controls: yes, including a solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):The concentration of DMF in the solvent control and all Cyclaprop treatment groups was 0.10 mL/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): During the course of the test, the appearance of the test solutions was observed in both the diluter mixing chambers, where test substance stocks and dilution water were combined prior to delivery to the test chambers, and in the test chambers. The test solutions in the mixing chambers and test chambers appeared clear and colorless during the test, with no evidence of precipitation observed in any control or treatment solution.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead Minnow
- Source: Chesapeake Cultures, Inc., Hayes, Virginia

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
Fathead minnow embryos used in the test were received at Wildlife International on spawning substrates. Upon receipt, the embryos were removed from the spawning substrates and examined under a dissecting microscope to select healthy, viable specimens at approximately the same stage of development. Embryos collected for use in the test were from 10 individual spawns and were <24 hours old when the test was initiated. To initiate the test, groups of 1 to 3 embryos were impartially distributed among incubation cups until each cup contained 20 embryos. One cup was placed in each treatment and control replicate.

POST-HATCH FEEDING
- Type/source of feed: live brine shrimp nauplii (Artemia sp.), obtained by hatching cysts purchased from Brine Shrimp Direct, Ogden, Utah.
- Amount given: Not specified. To ensure that the feeding rate per fish remained constant, rations were adjusted at least weekly to account for losses due to mortality.
- Frequency of feeding: Newly-hatched larvae were fed three times per day during the first seven days of post-hatch. Thereafter, they were fed three times per day on weekdays and at least two times per day on weekends. Fish were not fed for approximately 48 hours prior to the termination of the test to allow for clearance of the digestive tracts before weight measurements were made.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Remarks on exposure duration:

5 day hatching period and 28-day post-hatch juvenile growth period

Hardness:

141 ± 4 mg/L as CaCO3, moderately hard

Test temperature:

24.8 - 25.9°C

pH:

8.0 - 8.3

Dissolved oxygen:

4.9 - 8.2 mg/L

Nominal and measured concentrations:

Nominal concentrations: 0.31, 0.63, 1.3, 2.5 and 5.0 mg/L
Mean measured concentrations: 0.20, 0.38, 0.80, 1.5 and 3.4 mg/L (measured concentrations of test solution samples collected on Days 0, 6, 13, 20, 27 and 33 of the test, averaged for each treatment group), representing 65, 60, 62, 60, 68 % of nominal concentrations, respectively. Although the analytical results had recoveries below 80% of nominal, the results were consistent among all treatment concentrations throughout the study. The results of the study were based on the mean measured concentrations.

Details on test conditions:

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): Embryos were held in incubation cups constructed from glass cylinders approximately 50 mm in diameter with 425 µm nylon screen mesh attached to the bottom with silicone sealant. The cups were suspended in the water column of each test chamber and attached to a rocker arm. The reciprocating motion of the rocker arm (2 rpm) facilitated circulation of test water around the embryos during incubation.
- Test vessel: 9-L glass aquaria filled with approximately 7 L of test solution. The depth of the test water in a representative test chamber was 16 cm.
- Type of flow-through (e.g. peristaltic or proportional diluter): continuous-flow diluter
- Renewal rate of test solution (frequency/flow rate): The diluter flow rate was adjusted to provide approximately five volume additions of test water in each test chamber per day.
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: Biomass loading at the end of the test, based on the mean wet weight of the negative control group, was 0.057 g of fish per liter of test solution that passed through the test chamber during a 24-hour period. Instantaneous loading (the total wet weight of fish per liter of water in the tank) at the end of the test was 0.30 g fish/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International site. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800 L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- Metals:
Components and Measured Concentrations (mg/L)

Aluminum < 0.200
Antimony < 0.0200
Arsenic < 0.0200
Barium < 0.0050
Beryllium < 0.0050
Bromide < 2.5
Cadmium < 0.0050
Calcium 33.5
Chloride 4.2
Chromium < 0.0150
Cobalt < 0.0050
Copper < 0.0100
Fluoride < 0.50
Iron < 0.200
Lead < 0.0150
Magnesium 12.9
Manganese < 0.0050
Mercury < 0.00020
Nickel < 0.0100
Nitrate Nitrogen < 0.50
Nitrite Nitrogen < 0.50
Potassium 6.57
Selenium < 0.0200
Silver < 0.0050
Sodium 18.2
Sulfate < 5.0
Thallium < 0.0300
Vanadium < 0.0050
Zinc < 0.0200

- Pesticides and organics:

Components and Measured Concentrations (µg/L)

Aldrin < 0.0082
Alpha BHC < 0.0082
Alpha Chlordane < 0.0082
Beta BHC < 0.0082
Bolstar < 4.9
Chlordane < 0.41
Coumaphos < 4.9
Delta BHC < 0.0082
Demeton-O < 4.9
Demeton-S < 4.9
Diazinon < 4.9
Dichlorvos < 4.9
Dieldrin < 0.016
Disulfoton < 4.9
Dursban (Chlorpyrifos) < 4.9
Endosulfan I < 0.0082
Endosulfan II < 0.016
Endosulfan Sulfate < 0.016
Endrin < 0.016
Endrin Aldehyde < 0.082
Endrin Ketone < 0.016
EPN < 4.9
Ethion < 4.9
Ethoprop < 4.9
Ethyl Parathion < 4.9
Famphur < 4.9
Fensulfothion < 4.9
Fenthion < 4.9
Gamma BHC – Lindane < 0.0082
Gamma Chlordane < 0.016
Guthion (Azinphos-methyl) < 4.9
HCB < 0.0082
Heptachlor < 0.0082
Heptachlor Epoxide < 0.0082
Kepone < 0.16
Malathion < 4.9
Merphos < 4.9
Methoxychlor < 0.082
Methyl Parathion < 4.9
Mevinphos < 4.9
Mirex < 0.20
Naled < 4.9
o,p-DDD < 0.016
o,p-DDE < 0.61
o,p-DDT < 0.016
p,p-DDD < 0.016
p,p-DDE < 0.016
p,p-DDT < 0.016
Phorate < 4.9
Ronnel < 4.9
Stirophos < 4.9
Telodrin < 0.0082
Tokuthion < 4.9
Toxaphene < 2.4
Trichloronate < 4.9
Trithion < 4.9

- Alkalinity: Mean 177 (range 174 - 178) mg/L as CaCO3
- Conductivity: Mean 329 (range 298 - 355) µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was measured in each test chamber at the beginning of the test, weekly during the test, and at the end of the test using a liquid-in-glass thermometer. Dissolved oxygen and pH were measured in alternating replicates of each treatment and control group at the beginning of the test, weekly during the test, and at the end of the test. When 100% mortality occurred in the replicate, measurements of temperature, dissolved oxygen and pH were taken in that replicate and then discontinued.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting.
- Light intensity: Light intensity at test initiation was 210 lux at the surface of the water of one representative test chamber.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
During the first day of exposure, embryos were observed twice for mortality and eggs with fungus. Thereafter, until hatching was complete, observations of embryo mortality and the removal of dead embryos were performed once daily. When hatching reached >90% in the control groups on Day 5 of the test, the larvae were released to their respective test chambers and the post-hatch period began. Any unhatched embryos were kept in the egg cups until they hatched and were released into the test chamber, or until death of the embryo occurred. During the 28 day post-hatch exposure period, the larvae were observed daily to evaluate the numbers of mortalities and the numbers of individuals exhibiting clinical signs of toxicity or abnormal behavior. From these observations, time to hatch, hatching success, and post-hatch growth and survival were evaluated. Hatching success was calculated as the percentage of embryos that hatched successfully. Post-hatch survival was calculated as the number of larvae surviving to test termination divided by the total number of embryos that hatched successfully.
Post-hatch growth of the fathead minnows was evaluated at the conclusion of the 28-day post-hatch exposure period. Total length for each surviving fish was measured to the nearest 1 mm using a metric ruler, and wet and dry weights were measured to the nearest 0.1 mg using an analytical balance. Fish were placed in an oven at approximately 60°C for approximately 120 hours to obtain dry weight data.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 0.081, 0.27, 0.90, 3.0 and 10 mg/L
- Results used to determine the conditions for the definitive study:
The majority of fathead minnow embryos in the control and treatment replicates hatched on Days 4 and 5 of the test, with no apparent treatment related effect on time to hatch. Mean percent hatching success in the negative control, solvent control, 0.081, 0.27, 0.90, 3.0 and 10 mg/L treatment groups was 100, 100, 98, 100, 100, 98 and 84%, respectively. Mean percent survival in the negative control, solvent control, 0.081, 0.27, 0.90, 3.0 and 10 mg/L treatment groups was 94, 98, 94, 96, 90, 94 and 0%, respectively. Mean group wet weight in the negative control, solvent control, 0.081, 0.27, 0.90 and 3.0 mg/L treatment groups was 17.5, 21.3, 16.7, 17.2, 21.3 and 12.4 mg, respectively. Based on the results of the preliminary range-finding test, the nominal test concentrations of 0.31, 0.63, 1.3, 2.5 and 5.0 mg/L were selected in consultation with the Sponsor for the definitive test.

POST-HATCH DETAILS
- Begin of post-hatch period: When hatching reached >90% in the control groups on Day 5
- No. of hatched eggs (alevins)/treatment released to the test chamber: 20
- Release of alevins from incubation cups to test chamber on day no.: 5

Reference substance (positive control):

no

Key result

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: sublethal effects incl behaviour and morphological changes

Remarks on result:

other: LOEC = 1.5 mg/L

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth: total length, wet and dry weight

Remarks on result:

other: LOEC = 1.5 mg/L

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: larval survival

Remarks on result:

other: LOEC is 1.5 mg/L

Duration:

33 d

Dose descriptor:

other: MATC

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth

Duration:

33 d

Dose descriptor:

EC10

Effect conc.:

0.91 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: larval survival

Remarks on result:

other: 95% conf. int 0.049 - 1.06 mg/L

Duration:

33 d

Dose descriptor:

other: EC20

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: larval survival

Remarks on result:

other: 95% conf. int. 0.97 - 1.34 mg/L

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

3.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks on result:

other: Presented as EC10 > 3.4 mg/L

Details on results:

For details see table in field: 'any other information of results'
- Days to hatch: No apparent differences in time to hatch between control groups and any of the treatment groups. Majority in control and treatment replicates hatched on days 4 and 5. Hatching reached > 90% in control on day 5. A few inbryos in solvent control, 0.20, 0.38, 1.5 and 3.4 mg/L groups hatched or died on day 6.
- Numbers hatched: pooled control 97%; 0.20 mg/L 95%; 0.38 mg/L 94%; 0.80 mg/L 90%; 1.5mg/L 95%; 3.4 mg/L 90%. Although the groups with 90% hatching were statistically different from the pooled control (p≤ 0.5), it was stated that the reduction was not dose-response related and the hatching success still exceeded the control criterion, so the differences were not considered to be biologically meaningful or treatment related.
- Observations on body length and weight of alevins:
No statistically significant differences in any of the growth parameters between the negative and solvent control groups (p > 0.05), so the data were pooled for comparisons with the treatment groups. Dunnett’s one-tailed test indicated statistically significant reductions in total length and wet weight among fish in the 0.80 mg/L treatment group and statistically significant reductions in dry weight in the 0.20 and 0.80 mg/L treatment groups in comparison to the pooled controls (p ≤ 0.05). Since the reductions in growth at the 0.20 mg/L treatment group did not follow a dose response pattern, the reductions in growth at the 0.20 mg/L treatment group were not considered to be treatment related. In addition, the reductions in mean total length, wet weight and dry weight in the 0.80 mg/L treatment group from the pooled controls (3.2, 8.8 and 6.4%, respectively) were less than 10% and were not considered to be biologically meaningful. Consequently, the NOEC for growth was 0.80 mg/L and the LOEC was 1.5 mg/L.
- Sublethal effects:
The majority of the fish in the controls and in the 0.20, 0.38 and 0.80 mg/L treatment groups that survive to test termination appeared normal, with occasional incidental observations of small or weak fish or a fish with a crooked spine.
At the 1.5 and 3.4 mg/L test concentrations sublethal signs of toxicity included erratic swimming, loss of equilibrium and fish lying on the bottom of the test chambers. Fish that were weak or smaller in stature were also noted at a higher frequency of occurrence at these concentrations. The larvae in the 3.4 mg/L treatment group were noted lying at the bottom of the test chambers from Day 0 post-hatch until they were all dead on Day 5 post-hatch.

Summary of hatching succes, larval survival and growth

Mean Measured Concentration (mg/L)	Percent Hatching Success	Percent Survival to Day 28 Post-Hatch	Growth Parameters at Day 28 Post-Hatch
			Mean Total Length ± SD (mm)	Mean Wet Weight ± SD (mg)	Mean Dry Weight ± SD (mg)
Negative Control	98	97	24.7 ± 0.22	103.1 ± 3.2	20.5 ± 0.90
Solvent Control	96	99	24.8 ± 0.50	106.1 ± 8.4	20.2 ± 1.4
Pooled Control	97	98	24.7 ± 0.37	104.6 ± 6.1	20.3 ± 1.1
0.20	95	93^3	24.2 ± 0.40^3	97.0 ± 5.3^3	18.3 ± 1.0*, ^3, 5
0.38	94	93	24.6 ± 0.32	103.7 ± 6.2	20.3 ± 0.66
0.80	90*,^1	93	23.9 ± 0.42*^,6	95.4 ± 2.5*,^6	19.0 ± 0.42*,^6
1.5	95	70*	22.7 ± 0.83^4	89.4 ± 6.2^4	19.0 ± 1.5^4
3.4	90*,^2	0*	--	--	--
EC10 (95% CI)^4	>3.4	0.86 (0.049 – 1.1)	2.7 (0.37 – 20)	1.3 (0.15 - 12)	NC
EC20 (95% CI)^4	>3.4	1.2 (0.97 – 1.3)	6.7 (0.24 – 184)	5.1 (0.039 – 660)	NC
*            There were statistically significant reductions (p ≤ 0.05) in percent hatching success, survival and growth in comparison to the pooled controls (percent hatching success and survival using Fisher’s Exact test; growth using Dunnett’s one-tailed test). 1            Since the percent hatching success of the 0.80 mg/L treatment group did not follow a dose-response pattern the statistically significant reductions found in this treatment group in comparison to the pooled controls was not considered to be biologically meaningful. 2            Since percent hatching success at the 3.4 mg/L treatment group was comparable to the percent hatching success of the 0.80 mg/L treatment group and exceeded the control criterion of 70%, the statistically significant reduction found was not considered to be biologically meaningful. 3            Data from Replicate D of the 0.20 mg/L treatment group were excluded from the analysis of survival and growth since the mortality in this replicate occurred prior to the larvae being released into the growth chamber and was not considered to be related to treatment. 4            Data from the 1.5 mg/L treatment group were excluded from the analysis of growth end points due to significant effect on survival. 5            Since the mean dry weight of the 0.20 mg/L treatment group did not follow a dose-response pattern, the statistically significant reduction noted in mean dry weight at the 0.20 mg/L treatment group was not considered to be biologically meaningful. 6            Since the percent reductions in mean total length, wet weight and dry weight of the 0.80 mg/L treatment group from the pooled control were slight (3.2, 8.8 and 6.4%, respectively), the reductions noted were not considered to be biologically meaningful.  -- = No data due to 100% mortality.  NC = Not calculated. Results were overly wide.

Validity criteria fulfilled:

yes

Remarks:

Dissolved oxygen concentration was above 60% of the air saturation values throughout the test; the % succesfully hatched in the control groups were > 70% and post-hatch survival was > 75%.

Conclusions:

The 33d-NOEC of Cyclaprop in a fish early life-stage test with Pimephales promelas was 0.8 mg/L, based on growth and other sublethal effects. The LOEC was 1.5 mg/L and the MATC was 1.1 mg/L.

Executive summary:

An early life-stage toxicity test was performed with the fathead minnow (Pimephales promelas) according to OECD TG 210 (v. 2013) and in compliance with GLP standards. Fish eggs aged less than 24 h were exposed in a flow-through system for 33 days (5 days to hatching and 28 days post hatch). Dimethyl formamide was used to prepare the stock solutions. A solvent control was included. Nominal test concentrations were 0.31, 0.63, 1.3, 2.5 and 5.0 mg/L whereas the mean measured concentrations were 0.20, 0.38, 0.80, 1.5 and 3.4 mg/L, respectively. These levels represented between 60 and 68% of the nominal concentrations. There were no signficant differences between the effects in the control and the solvent control and so the data were pooled.

The results are based on the mean measured concentrations.

No significant treatment-related effects on survival were noted at concentrations up to and including 3.4 mg/L, whereas survival was affected starting at 1.5 mg/L. Sublethal effects (behaviour and morphology) were seen starting at a concentration of 1.5 mg/L and above.

Growth (measured as total length, wet and dry weight), larval survival and sublethal effects were the determining endpoint with 33d-NOEC = 0.8 mg/L. The LOEC was 1.5 mg/L and the MATC was 1.1 mg/L. The lowest 33d-EC10 value was 0.86 (0.049 - 1.1) mg/L for survival; the 33d-EC20 value was 1.2 (0.97 - 1.3) mg/L.

Reference 2

Endpoint:

long-term toxicity to fish, other

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Information is derived from the analogue Cyclaprop

Justification for type of information:

The full read across justification is presented in the Aquatic toxicity Endpoint summary. The value for Cyclabute is converted using the acute / chronic ratio from Cyclaprop.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

33 d

Dose descriptor:

EC10

Effect conc.:

0.34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Acute / Chronic ratio

Validity criteria fulfilled:

yes

Remarks:

Annex XI Read across justification is met

Conclusions:

Cyclabute has an EC10 for long-term fish toxicity of 0.34 mg/l based on the acute/chronic ratio of Cyclaprop fish toxicity

Executive summary:

Cyclabute's long term fish toxicity is derived from acute fish toxicity of Cyclobutanate, which is 15.8 mg/l and the acute / chronic ratio of Cyclaprop, which is 8.4. This results in a long-term fish toxicity value of 1.9 mg/l. A full justification is copied into the Endpoint summary.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.43 mg/L

Additional information

The long term fish toxicity is derived from the acute fish toxicity of Cyclobutanate and the derived acute / chronic ratio of Cyclaprop. The executive summary of the long term study of Cyclaprop is presented here. The read across justification and the A/C ratio application justification is presented in the Aquatic toxicity Endpoint summary.

Cyclaprop and its long-term fish toxicity

An early life-stage toxicity test was performed with the fathead minnow (Pimephales promelas) according to OECD TG 210 (v. 2013) and in compliance with GLP standards. Fish eggs aged less than 24 h were exposed in a flow-through system for 33 days (5 days to hatching and 28 days post hatch). Dimethyl formamide was used to prepare the stock solutions. A solvent control was included. Nominal test concentrations were 0.31, 0.63, 1.3, 2.5 and 5.0 mg/L whereas the mean measured concentrations were 0.20, 0.38, 0.80, 1.5 and 3.4 mg/L, respectively. These levels represented between 60 and 68% of the nominal concentrations. There were no signficant differences between the effects in the control and the solvent control and so the data were pooled.

The results are based on the mean measured concentrations.

No significant treatment-related effects on survival were noted at concentrations up to and including 3.4 mg/L, whereas survival was affected starting at 1.5 mg/L. Sublethal effects (behaviour and morphology) were seen starting at a concentration of 1.5 mg/L and above.

Growth (measured as total length, wet and dry weight), larval survival and sublethal effects were the determining endpoint with 33d-NOEC = 0.8 mg/L. The LOEC was 1.5 mg/L and the MATC was 1.1 mg/L. The lowest 33d-EC10 value was 0.86 (0.049 - 1.1) mg/L for survival; the 33d-EC20 value was 1.2 (0.97 - 1.3) mg/L.