Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Nov 2006 to 15 Dec 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted by the Council 17th July 1992

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

Public Draft, (April 1996)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals: Analytical samples were taken from the test concentrations and controls on days 0, 7, 14, 21, 28 and at the end of the test (day 32).
- Sampling procedure: On each sampling occasion, duplicate samples of approximately 100 mL were taken from the centre of each replicate test vessel sampled. At the start and end of the study one sample from all replicate treatments and controls was analysed. On other days only one set of replicates A, B or C was analysed. For example on day 7 replicate A was analysed, and replicate B on day 14 etc. Other replicate samples, and all duplicates were stored. Samples of the application solution (5 mL) were taken periodically.

Vehicle:

yes

Remarks:

tetrahydrofuran

Details on test solutions:

- Controls: Dilution water control and a solvent control containing 50 µL of tetrahydrofuran (THF) per litre of dilution water.
- Method of preparation of the application solution: A concentrated aqueous application solution was prepared by adding 80 mg of the test substance to 1 L of a mixture of THF and deionised water (25:75% v/v) to give an 80 mg/L solution. The application solution was then diluted to provide the test concentration series, with the solvent control and test concentrations containing 50 µL of THF per litre of dilution water. The application solution was renewed weekly. Application solution consumption was monitored by weighing the application solution bottle on a daily basis.
- Vehicle solution: The vehicle solution was 25% THF : 75% deionised water.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Feeding during test : Larvae were fed live brine shrimp (Artemia) and ZM100 larva food. From day 18 the daily diet was supplemented with finely ground flake fish food. Surplus food and waste was removed from the test vessels as necessary.
- Frequency: Twice daily from Day 5. Feeding was withheld for 24 hours prior to the measurements at study termination.

ACCLIMATION
- Acclimation conditions: The fertilised eggs were spawned and dispatched via air freight to the test facillity. The water temperature of the dilution water containing the eggs was 18°C. Therefore temperature equilibration time was needed. The eggs were pooled by removing the excess water and gently pouring them in a chamber filled with dilution water at 20.0°C. The water bath was adjusted to approximately 23°C on Day 0 to help acclimatise the eggs before increasing the temperature to 25°C on Day 1.
- Test organism handling: Eggs and newly hatched larvae were handled with wide bore pipettes. At test termination a fine-mesh dip net was used to transfer the fish.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Remarks on exposure duration:

4 days egg incubation + 28 days post-hatch

Hardness:

205 - 244 mg/L as CaCO3

Test temperature:

24.4 - 24.9 ˚C

pH:

7.45 - 8.10

Dissolved oxygen:

71 to 100 %ASV (air saturation value)

Conductivity:

470 - 490 μS/cm

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative contorl), 0 (solvent contril), 0.7, 1.5, 3.3, 7.3 and 16 µg/L.
- Measured concentrations: ND, ND, 0.52, 1.43, 2.87, 5.49 and 13.0 µg/L, respectively.

Details on test conditions:

TEST SYSTEM
- Egg cups: Glass cylinders (diameter approximately 7 cm)
- Type: Closed (at one end by fine stainless steel mesh net)
- No. of eggs per cup: 30
When the assignment of eggs was complete the egg cups were placed in the appropriate test vessel.
- Test vessel: All-glass 10 L aquaria
- Filled volume: Approximately 5 to 6 litres
- Aeration: From day 18 the test vessels were aerated gently using glass Pasteur pipettes, to ensure that the concentration of dissolved oxygen present in the vessels did not fall below 60% of the air saturation value (ASV).
- Type of flow-through: modified proportional diluter
- Flow rate: Approximately 12 media renewals per vessel every 24 hrs. Every diluter cycle delivered 0.5 L of water or test solution to one replicate. The diluter was set to run a cycle every 4 minutes, therefore, approximately 360 cycles were completed per 24 hours. As the diluter sequentially supplied the three replicates, over any 24-hour period each replicate received the volume delivered by 120 cycles, which corresponds to 60L of water or test solution per 24 hours.
- No. of eggs per vessel: 30
- No. of vessels per concentration: 3
- No. of vessels per control: 3
- No. of vessels per vehicle control: 3
- Biomass loading rate: The instantaneous fish loading in each replicate, based on a tank volume of 5 litres and the total wet weights of surviving fish on day 32, was calculated for each replicate.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was prepared by mixing mains water with deionised water to give a total hardness of 100 - 250 mg CaCO3/L. The mixed water was then passed through an activated carbon filter and a UV sterilizer to produce the dilution water. Analysis of representative samples of dilution water is conducted on at least a biannual basis for potential toxicants, pesticides, particulate matter, total organic carbon and metals. The results of the most recent analysis indicated that the dilution water was of a suitable quality, in agreement with the testing guidelines followed.
- Intervals of water quality measurement: Temperature, dissolved oxygen and pH measurements were recorded at test initiation and twice per week in all control and treatment replicates. One dilution water control vessel (replicate A) was monitored for temperature continuously. On day 0, the temperature of the water bath was adjusted to 23˚C to avoid thermal shock of the eggs and then increased to 25˚C the following day. Hardness and conductivity were measured alternately in each replicate dilution water control vessel at test initiation, once per week during the experimental phase, and at test end.

OTHER TEST CONDITIONS
- Photoperiod: A 16-hour light 8-hour dark photoperiod was maintained with a 30 minute dawn/dusk transition period.

EFFECT PARAMETERS MEASURED
- Egg viability assessment: Two sub-samples (2 x 50) of the remaining eggs were impartially selected and transferred to a 10% (vlv in dilution water) acetic acid solution. After some minutes the eggs became clear and it was therefore possible to count the number of eggs showing a white line (neural keel). The presence of a neural keel indicates the egg had been fertilized. The egg viability for the batch was calculated as the number of embryos with a neural keel out of the total number of eggs sampled, and is presented as a percentage.
- Hatching: Hatching was visually estimated daily, together with observations of egg mortality, determined by a distinct change in colouration (opalescent white). To avoid physical damage to adjacent live eggs, white eggs were not removed. On day 5 hatching was estimated to be 100% for the control vessels, with no un-hatched viable eggs remaining. Hence, day 5 was defined as "hatching day", i.e. the day when the highest number of larvae hatched in the control, and the larvae from all replicates were counted and transferred into the test vessels. White eggs were discarded at this point. The test was continued for 28 days post-hatch.
- Survival: Daily observations (visual inspection) of eggs and larval mortality, behaviour and appearance were made and any abnormal effects recorded. Actual counts of eggslfish occurred at test initiation (Day 0), when the larvae were released into the test vessels (Day 5), and at the test end (Day 32).
- Test end: At test termination, all surviving fish in each replicate were terminally anaesthetized using a Schedule 1 method as described in the Animals (Scientific Procedures) Act 1986. Juvenile fish were then measured for total length before being dried at 60°C for at least 24 hours and their dry weights determined.

Reference substance (positive control):

no

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

2.9 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: fry survival

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

5.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Overall survival, length, dry weight

Details on results:

An overview of the results is provided in Table 1 - Table 5 in 'Any other information on results incl. tables'
Pre-hatch phase
- Egg fertility: Fertile eggs are defined as the number of embryos with a neural keel out of the total number of eggs sampled, expressed as a percentage. The fertility of the batch of eggs used in the study was 92%, demonstrating the eggs were of a suitable quality for the test.
- Egg survival: Non-viable (dead) eggs are defined as those which turned white and failed to hatch. There was a statistically significant difference in egg viability between the dilution water control and solvent control at day 5. Hence, the subsequent statistical comparison for egg survival between treatment and control replicates utilised the solvent control data only. There was no statistical difference in egg viability between the solvent control and any of the test treatments. At the highest concentration (13.0 µg/L) mean egg viability was similar to the solvent control (89% and 94% respectively). However, embryonic development, estimated from the presence of eyed eggs (i.e. dark eyes becoming visible through the chorion), was delayed by approximately one day at 13.0 µg/L. By day 3, with the exception of 13.0 µg/L, developing eyes were seen in the eggs in all replicates of all treatments and controls. Eyed eggs were present in the 13.0 µg/L replicates on day 4.

Post hatch phase
- Hatching success: Mean hatching success in the dilution water control was 78%. In the solvent control this was 80%. As there was no statistically significant difference in hatching success between the dilution water control and the solvent control, the control data were pooled for subsequent analysis. There was no statistically significant difference between the pooled controls and any treatment as regards hatching success.
- Fry survival, days 5 – 32: Mean survival of fry, from day 5 to day 32, in the dilution water control was 81%. In the solvent control mean fry survival was 85%. As there was no statistically significant difference in fry survival between the dilution water control and the solvent control, the control data were pooled for subsequent analysis. At 13.0 µg/L, the highest concentration of the test substance tested, no fry survived to the end of the test. Hence, there was a statistically significant difference between the pooled controls and this treatment as regards fry survival. Statistical analysis of the remaining treatments identified significant reductions in fry survival, compared with the pooled controls, at 5.49 µg/L and 1.43 µg/L, but not at 2.87 µg/L or 0.52 µg/L. The Lowest Observed Effect Concentration for fry survival, (i.e. the 32-day LOECfry survival), defined as the lowest tested concentration of a test substance at which the test substance is observed to have a significant effect (p < 0.05) when compared to the appropriate control, and all tested concentrations above have a harmful effect equal to or greater than those observed at the LOEC, is 5.49 µg/L. The No Observed Effect Concentration for fry survival (i.e. the 32-day NOECfry survival), defined as the concentration immediately below the LOECfry survival, is 2.87 µg/L. The instantaneous fish loading rate in the dilution water control vessels at the end of the test ranged from 0.72 to 1.00 g/L, while in the solvent control vessels this was 0.64 to 0.80 g/L.
- Overall survival, days 0 - 32: Overall survival, from day 0 to day 32, in the dilution water control was 63%. In the solvent control overall survival was 68%. As there was no statistically significant difference in overall survival between the dilution water control and the solvent control, the control data were pooled for subsequent analysis. At 13.0 µg/L, the highest concentration of the substance tested, no fry survived to the end of the test. Hence, there was a statistically significant difference between the pooled controls and this treatment as regards overall survival at the end of the test. No significant differences in overall survival were found between the pooled controls and the remaining test treatments.
The 32-day LOECoverall survival was 13.0 µg/L.
The 32-day NOECoverall survival was 5.49 µg/L.

Sub-lethal effects
Hatching started synchronously in the controls and treatments on day 4. With the exception of the highest concentration of the substance tested (13.0 µg/L), this was complete by day 5, at which point the larvae were released from the egg cups and counted into the test vessels. Hence, day 5 was the hatching day. At 13.0 µg/L there were two eyed eggs (viable but un-hatched) present in each replicate upon release of the larvae into the test vessels. At this treatment, hatching was completed by day 6 as no eyed eggs or white eggs were present in these replicates at this time. A low incidence of larval spinal deformity was evident during the first 2.5 weeks of the study. Two types of spinal deformity were apparent during this phase:
- Larval scoliosis: When viewed from above, the spines of affected larvae were bent to approximately 45˚ in the lateral plain, at a distance of approximately 113 of the body length behind the head. The numbers of larvae affected were minimal (7%, i.e. 36 of 530 that hatched), and there was no relationship between this condition and the concentration of the test substance.
- Spinal curvature: A general curvature of the spine downwards throughout its length. This condition was dose related, as most of the larvae in the highest concentration of the substance tested (13.0 µg/L), i.e. 65 of 78 that hatched (83%), were affected by this form of spinal deformity which was absent from the other treatments and the controls. Larvae exhibiting both these types of spinal deformity failed to grow, probably due to an inability to feed effectively. Consequently, all those affected by both these conditions had died by day 17. A third type of spinal deformity, lordosis, became apparent as the test progressed. This was characterized by multiple spinal distortions in the dorso/ventral plain, such that affected spines had a zig-zag appearance when viewed from the side. This deformity was first noted on day 16 in replicate B of the dilution water control and in replicate A of the 2.87 µg/L dose. On day 18, one fish in replicate A at 1.43 µg/L was found to be similarly affected. By the end of the test the numbers of fish affected by this condition were 4 controls, 1 solvent control, 3 at 0.52 µg/L, 1 at 1.43 µg/L, 4 at 2.87 µg/L and 0 at 5.49 µg/L. There was no relationship between lordosis and the concentration of the test substance the fry were exposed to. All such affected fish survived to the end of the test but did not recover from this condition.

- Length: Mean total length (mm, ± standard deviation for the means of three replicates) for the dilution water control was 25.2 (± 1.5). For the solvent control this was 23.7 (±0.1). The controls were pooled prior to analysis (since p = 0.1619). The assumption of homogeneity of variance appears reasonable and Shapiro-Wilks test was not significance at the 5% level (p = 0.0771). None of the test item treatments were statistically significantly different from the pooled control at the 5% level. Therefore:
The 32-day LOEClength is 13.0 µg/L
The 32-day NOEClength is 5.49 µg/L

Dry weight: Mean dry weight (mg, ± standard deviation for the means of three replicates) for the dilution water control was 53.15 (±6.4). For the solvent control this was 40.66 (±1.7). The controls were statistically significantly different to each other (p = 0.0288), hence comparisons of dry weight were made with the solvent control. The assumption of homogeneity of variance appears reasonable and Shapiro-Wilks test was not significant at the 5% level (p = 0.5362). The 0.52 µg/L and 2.87 µg/L treatments were found to be statistically significantly different from the solvent control at the 5% level (p = 0.0203 and 0.0187 respectively). However, examination of these differences showed an increase in dry weight relative to the solvent control which is not considered to be an adverse effect. Therefore:
The 32-day NOECdry weight is 5.49 µg/L
The 32-day LOECdry weight is 13.0 µg/L.

Table 1. White eggs and hatched larvae during days 0 - 5

Day no.	Control						Solvent control
	Replicate A		Replicate B		Replicate C		Replicate A		Replicate B		Replicate C
	white	hatched	white	hatched	white	hatched	white	hatched	white	hatched	white	hatched
0	0	0	0	0	0	0	0	0	0	0	0	0
1	0	0	0	0	0	0	1	0	0	0	0	0
2	0	0	0	0	0	0	1	0	0	0	0	0
3	0	0	0	0	0	0	1	0	0	0	0	0
4	0	3	0	24	0	21	1	25	0	28	0	26
5*	6	23	4	22	5	25	0	24	2	25	3	23

 

	0.52 µg/L						1.43 µg/L
	Replicate A		Replicate B		Replicate C		Replicate A		Replicate B		Replicate C
	white	hatched	white	hatched	white	hatched	white	hatched	white	hatched	white	hatched
0	0	0	0	0	0	0	0	0	0	0	0	0
1	1	0	1	0	0	0	0	0	0	0	0	0
2	2	0	1	0	0	0	0	0	0	0	2	0
3	2	0	1	0	0	0	0	0	0	0	2	0
4	2	26	1	24	0	24	0	23	0	25	2	27
5*	3	22	4	20	1	24	2	25	3	26	4	26

 

	2.87 µg/L						5.49 µg/L
	Replicate A		Replicate B		Replicate A		Replicate B		Replicate A		Replicate B
	white	hatched	white	hatched	white	hatched	white	hatched	white	hatched	white	hatched
0	0	0	0	0	0	0	0	0	0	0	0	0
1	0	0	0	0	0	0	0	0	0	0	2	0
2	3	0	0	0	2	0	0	0	0	0	1	0
3	2	0	0	0	1	0	0	0	0	0	2	0
4	2	27	0	29	1	26	0	22	0	26	2	22
5*	4	26	0	29	4	25	2	28	1	29	2	25

 

	13.0 µg/L
	Replicate A		Replicate B		Replicate A
	white	hatched	white	hatched	white	hatched
0	0	0	0	0	0	0
1	0	0	0	0	0	0
2	0	0	0	0	0	0
3	0	0	1	0	2	0
4	0	0	1	5	2	3
5*	3+	28-	0	28-	8	22-

* = transfer day

+ one of these "dead eggs" may have been the opaque shell from a hatched egg

 - totals include two unhatched viable eggs each

Note: On day 0,30 eggs were placed in each of 21 egg cups and one egg cup was then assigned to each test vessel.

Numbers on days 0 to 4 are visual estimations.

Numbers on day 5 are actual counts made when the larvae were released from the egg cups into the vessels.

Missing organisms are assumed to have died shortly after hatching and rapidly decomposed.

Table 2. Summary of egg viability, hatching success, fry survival and overall survival 

Mean measured concentration	Replicate	Egg viability 1 (%)	Hatching success2 (%)	Larval survival 3 (%)	Overall surviva l4 (%)
µg/L
Control	A	80	77	78	60
	B	87	73	86	63
	C	83	83	80	67
	Mean	83	78	81	63
Solvent control	A	100	80	92	73
	B	93	83	84	70
	C	90	77	78	60
	Mean	94	80	85	68
0.52	A	90	73	91	67
	B	87	67	95	63
	C	97	80	96	77
	Mean	91	73	94	69
1.43	A	93	83	64	53
	B	90	87	77	67
	C	87	87	58	50
	Mean	90	86	66	57
2.87	A	87	87	77	67
	B	100	97	97	93
	C	87	83	76	63
	Mean	91	89	83	74
5.49	A	90	93	93	87
	B	97	97	52	50
	C	93	83	44	37
	Mean	93	91	63	58
13.0	A	93	93	0	0
	B	100	93	0	0
	C	73	73	0	0
	Mean	89	86	0	0

1 the number of eggs at the start of the test (day 0) minus the number of dead eggs, expressed as a percentage of the number of eggs added at the start of the test (day 0).

2 the number ofl & larvae on the day they are transferred from the egg cups to the test vessels (day 5), expressed as a percentage of the number of eggs added at the start of the test (day 0).

3 the number of surviving larvae at end of test (day 32) expressed as a percentage of the number of live larvae on day 5.

4 the number of surviving larvae at end of test (day 32) expressed as a percentage of the number of eggs added on day 0.

Table 3. Effects of the test substance on the growth of Pimephales promelas

Nominal concentration (µg/L)	Mean measured concentration (µg/L)	Hatching success (%) 1	Quantal responses		Non quantal responses
			Fry survival day 5 to test end (%) 2	Overall survival day 0 to test end (%) 3	Mean length (mm) ± SD	Mean dry weight (mg)
Control	Control	78	81	63	25.2 ± 1.5	53.15 ± 6.4
Solvent control	Solvent control	80	85	68	23.7 ± 0.1	40.66 ± 1.7
0.7	0.52	73	94	69	25.3 ± 0.7	50.35 ± 5.4
1.5	1.43	86	66	57	24.6 ± 0.5	47.36 ± 1.5
3.3	2.87	89	83	74	25.4 ± 0.5	50.77 ± 4.0
7.3	5.49	91	63	58	23.4 ± 0.7	37.35 ± 3.0
16	13	86	0	0	-	-

1 the number of live larvae on the day they are transferred from the egg cups to the test vessels (day 5), expressed as a percentage of the number of eggs added at the start of the test (day 0).

2 the number of surviving larvae at the end of the test (day 32), expressed as a percentage of the number of live larvae on day 5.

3 the number of surviving larvae at the end of the test (day 32), expressed as a percentage of the number of eggs added on day 0.

Validity criteria

The study is considered valid because:

- Dissolved oxygen levels: Dissolved oxygen concentrations were maintained between 71 and 100% air saturation throughout the study (required ≥ 60%).

- Temperature: Temperature was maintained at 25 + 1°C throughout the test. The maximum difference in water temperature between test vessels, and between successive days, was 0.5 and 0.5˚C respectively, with the exception of the first 24 hours (required no more than +/- 1.5˚C).

- Control survival: Hatching success and fry survival (day 5 to 32) in the dilution watercontrol were 78% and 81 % respectively (required equal to or greater than 66% and 70% respectively). For the solvent control these values were 80% and 85% respectively.

- Solubilising agent effects: No adverse effects due to the solubilising agent, tetrahydrofuran, on the early-life stages of fathead minnow were detected during the test.

Validity criteria fulfilled:

yes

Remarks:

See validity criteria in 'Any other information on results incl. tables'

Conclusions:

In the early life stage toxicity test according to OECD TG 210, the 32-day NOEC for fry survival in fathead minnow (Pimephales promelas) was determined to be 2.9 µg/L, based on mean measured concentrations.

Executive summary:

The early-life stage toxicity of the substance was performed on fathead minnow (Pimephales promelas). The study was carried out in accordance with OECD TG 210 and OPPTS 850.1400, and in compliance with GLP. The fertilised egg (30 eggs/ test vessel; 3 vessels/ concentration or controls) was exposed to a range of nominal concentrations of 0.7, 1.5, 3.3, 7.3 and 16 μg/L, a solvent control (tetrahydrofuran) and a dilution water control for 32 days (28 days post-hatch). The mean measured concentrations were 0.52, 1.43, 2.87, 5.49 and 13 μg/L. The test conditions were  24 - 25 ˚C, pH 7.45 – 8.10, dissolved oxygen 71 – 100% air saturation value (ASV) and 16 / 8 hours light/dark cycle with 30 mins transition periods.

The results show that egg survival and hatching success was unaffected at all concentrations of the substance tested. There was delayed embryonic development and hatching at 13 μg/L and 100% mortality by day 14. Statistical analysis of the lower treatments levels identified significant reductions in fry survival, compared with the pooled controls, at 5.49 µg/L and 1.43 µg/L, but not at 2.87 µg/L or 0.52 µg/L. From day 0 to day 32, no significant differences in overall survival were found between the pooled controls and the remaining test treatments. None of the test item treatments was statistically significantly different in length or dry weight from the pooled control at the 5% level. Based on the findings, the 32-day NOEC for overall survival, length and dry weight was 5.49 μg/L. The lowest NOEC was the 32-day NOEC fry survival, 2.9 μg/L, based on mean measured concentrations.

Description of key information

Freshwater, 32-d NOEC = 2.9 µg/L (based on mean measured concentrations), fry survival, Pimephales promelas, OECD TG 210 and OPPTS 850.1400 guidelines, Sim 2007

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

2.9 µg/L

Additional information

One early life stage toxicity study is available for this endpoint, which was selected as the key study. In this study performed according to OECD TG 210 and OPPTS 850.1400 guidelines, and in compliance with GLP, fertilised fish egg (Pimephales promelas; 30 eggs / test vessel; 3 vessels/ concentration or controls) were exposed to the test substance at concentrations of 0.7, 1.5, 3.3, 7.3 and 16 μg/L, a solvent control (tetrahydrofuran) and a dilution water control for 32 days (28 days post-hatch). The mean measured concentrations were 0.52, 1.43, 2.87, 5.49 and 13 μg/L. The test conditions were 24 - 25 ˚C, pH 7.45 – 8.10, dissolved oxygen 71 – 100% ASV and 16 / 8 hours light/dark cycle with 30 mins transition periods. The results show that egg survival and hatching success were unaffected at all concentrations of the substance tested. There was delayed embryonic development and hatching at 13 μg/L and 100% mortality by day 14. The 32-day NOEC for fry survival (i.e., post-hatch until end of the test) was determined to be 2.9 μg/L, based on mean measured concentrations.