spirodiclofen (ISO);3-(2,4-dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutyrate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This toxicity study is classified as acceptable and satisfies the guideline requirement for early life toxicity study with fish.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 72-4 (Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM 1241-92

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The 97-day chronic toxicity of spirodiclofen technical to early life stage of rainbow trout (Oncorhynchus mykiss) was studied under flow-through conditions. Fertilized eggs (35 eggs per replicate for 140 eggs per test level; eggs were <24 hours old at start of exposure) of rainbow trout were exposed to control, solvent control (acidified methanol), and nominal (measured) test chemical concentrations of 0.123 (0.112), 0.25 (0.29), 0.49 (57), 0.98 (1.09), 1.97 (1.95), and 3.93 (3.81) µg/L. The parameters measured in this study were egg hatchability, survival, and fry growth, expressed by different toxic endpoints (egg hatch, time to hatch, time to swim-up, fry survival, fry growth (length and weight), morphological and behavioral effects).
Egg mortality: observed working daily during incubation period, dead eggs were discarded
No. eggs hatched: Day 39
Mortality of fry: Day 39 and daily thereafter
Swim-up behavior: Day 39 and daily thereafter
Growth measurements: study termination (Day 39); on individual fish - standard length, dry weight, wet weight (only on controls and solvent controls)
Embryonic development: not specifically reported but monitored through egg mortality observations (working daily)
Other sublethal effects: Day 39 and daily thereafter

Vehicle:

yes

Details on test solutions:

- Method: Continuous flow diluter using Prominent micro g/5 pumps for the intermittent introduction of stock solutions into corresponding water streams. There were six treatment levels plus a control and solvent control (acidified methanol). Flow splitting accuracy was within the prescribed 10% as determined prior to test initiation and after test termination. The diluter system and syringe pump
function were checked each working day. Aeration was not used to mix. The flow rate was set at 15 L/hour/rep which resulted in approximately 24 changes of test water per day.
- Controls: Control, solvent control and treatment levels
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Methanol (acidified with
2.5% glacial acetic acid to minimize degradation of Spirodiclofen in stock solutions). The solvent load was 0.1 mL/L.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Species: Rainbow trout (Oncorhynchus mykiss)
Age /embryonic stage at test initiation: Less than 24 hours (approximately 1-2
hours old before distribution to egg cups)
Method of collection of the fertilized eggs: Unfertilized rainbow trout eggs and milt for this study were obtained from a certified disease free commercial hatchery. These eggs and milt were collected from 3 female and 3 male adult brood fish. The unfertilized eggs and milt were acclimated from a transport temperature of 6°C to a temperature of 10°C over a period of 60 minutes (1°C/15 min) in the laboratory. Milt, transported in a separate plastic bag, was examined under the microscope. There were no negative findings. Upon acclimation, eggs were transferred to a dry stainless steel bowl and the milt was mixed with the eggs (with an autoclaved goose feather) for about 20 seconds. Dilution water (10±1°C) was added until the eggs were covered by 1 to 2 cm. The eggs and milt were gently stirred for about 1 minute to facilitate fertilization. After this stirring and an additional waiting period of about 5 minutes without stirring the eggs were gently rinsed with 10±1°C dilution water until the milt was no longer visible in the rinse water. The eggs were allowed to water harden for approximately 1-2 hours before distribution to incubation cups in the test system.
Source: Forellenzucht Worbis, Leinefelde, FRG.

Eggs and milt were purchased from a certified disease-free commercial hatchery so no parental acclimation was done at the laboratory.

Test type:

flow-through

Water media type:

freshwater

Total exposure duration:

97 d

Hardness:

Hardness: 45-52 mg/L as CaCO3

Test temperature:

Temperature: 9.8-11.0°C

pH:

pH: 6.4-7.3

Dissolved oxygen:

DO: 94-104%

Nominal and measured concentrations:

Nominal: Control, Solvent
Control, 0.123, 0.25, 0.49, 0.98,
1.97, and 3.93 µg/L.
Measured: <0.07, <0.07, 0.11,
0.29, 0.57, 1.09, 1.95, and 3.81
µg/L.

Details on test conditions:

Test System:
- Emybro cups (if used, type/material, size, fill volume): Eggs were incubated in incubation cups, constructed from 8 cm diameter teflon pipes with stainless steel plates perforated with holes (hole diameter: 1.8 mm) on the bottom. These incubation cups were suspended in each replicate test chamber. There is no specified fill volume as cups are suspended in test vessels.
- Test vessel: The glass aquaria used for testing measured approximately 120 mm x 143 mm with a water depth of 210 mm, yielding an approximate chamber volume of 3.6 liters.
- Aeration: There was no aeration during the test. Dilution water was aerated prior to introduction to test material and the experimental system.
- Type of flow-through (e.g. peristaltic or proportional diluter): Continuous flow diluter using Prominent micro g/5 pumps for the intermittent introduction of stock solutions into corresponding water streams.
- Renewal rate of test solution (frequency/flow rate):The flow rate was set at 15 L/hour/rep which resulted in approximately 24 changes of test water per day.
- No. of fertilized eggs/embryos per vessel: There were 35 fertilized eggs/egg incubation cup.
There were 4 replicates per test level. There were 140 fertilized eggs/test level. 4 replicates for control, solvent control, and all treatment levels

Water Parameters:
- Source/preparation of dilution water: Reconstituted water prepared by adding salt stock solutions to demineralized water to yield the desired nominal ionic concentrations.
- Conductivity: 85.6-111.8 µS/cm
- Alkalinity: 8-12 mg/L as CaCO3
- Intervals of water quality measurement: Temperature was monitored hourly. DO, pH, hardness, and alkalinity were measured approximately weekly. Conductivity was measured hourly.
- Photoperiod: Photoperiod: 16 hour light/8 hour dark
- Solvent: Methanol (acidified with 2.5% glacial acetic acid to minimize degradation of spirodiclofen in stock solutions). The solvent load was 0.1 mL/L.

Range-finding study: A range-finding study was conducted. Nominal concentrations for the definitive study were based on this test. The results of the study were not reported other than an effect threshold of between 1 and 5 µg/L based on effects of growth was established.

Post-hatch details:
The post hatch period began on Day 36. On Post Hatch Day (PHD) 3 (study day 39) the alevin were thinned to 15 individuals per replicate (60 individuals per test level). The hatch success in each replicate of the control and solvent control ranged from 89 - 100%.

Post-hatch feeding:
Feeding began on day 48 (posthatch day 12). Fry were fed ad libitum. Food was added to the aquaria twice daily, except on weekends/holidays when food was added once daily. Live brine shrimp nauplii were fed to the fry until one day before study termination. The brine shrimp cysts were purchased from Aquarium Products of Glen Burnie, Maryland, U.S.A. The brine shrimp cysts were analyzed for heavy metal and pesticide contamination prior the use and met ASTM recommendations.

Fertilization success study: Four replicates, each with 50 eggs in separate egg incubation cups were placed in exposure chambers with dilution water alone for determination of fertilization success. Egg fertilization rate was checked 12 days after fertilization with these eggs. The fertilization success in the four replicates ranged from 94 to 100 percent with a mean of 96.5 percent.

Observations: The parameters measured in this study were egg hatchability, survival, and fry growth, expressed by different toxic endpoints (egg hatch, time to hatch, time to swim-up, fry survival, fry growth (length and weight), morphological and behavioral effects).

Water quality was acceptable : Yes
Were raw data included? Yes

Reference substance (positive control):

no

Key result

Duration:

97 h

Dose descriptor:

LOEC

Effect conc.:

> 3.81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Key result

Duration:

97 d

Dose descriptor:

NOEC

Effect conc.:

3.81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Key result

Duration:

97 d

Dose descriptor:

EC50

Effect conc.:

> 3.81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Key result

Duration:

97 d

Dose descriptor:

LOEC

Effect conc.:

> 3.81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

morphology

Key result

Duration:

97 d

Dose descriptor:

LOEC

Effect conc.:

3.81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

length and weight; Day 97

Key result

Duration:

55 d

Dose descriptor:

LOEC

Effect conc.:

> 3.81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

time to swim up

Remarks:

(Day 55)

Key result

Duration:

39 d

Dose descriptor:

LOEC

Effect conc.:

> 3.81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

time to hatch

Remarks:

(Day 39)

Key result

Duration:

39 d

Dose descriptor:

LOEC

Effect conc.:

> 3.81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

number hatched

Remarks:

(Day 39)

Key result

Duration:

97 d

Dose descriptor:

LOEC

Effect conc.:

> 3.81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

other: fry survival

Key result

Duration:

97 d

Dose descriptor:

NOEC

Effect conc.:

> 3.81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

morphology

Key result

Duration:

97 d

Dose descriptor:

NOEC

Effect conc.:

1.95 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

length and weight; Day 97

Key result

Duration:

55 d

Dose descriptor:

NOEC

Effect conc.:

>= 3.81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

time to swim up

Remarks:

(Day 55)

Key result

Duration:

39 d

Dose descriptor:

NOEC

Effect conc.:

>= 3.81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

number hatched

Remarks:

(day 39)

Key result

Duration:

39 d

Dose descriptor:

NOEC

Effect conc.:

>= 3.81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

time to hatch

Remarks:

(day 39)

Key result

Duration:

97 d

Dose descriptor:

NOEC

Effect conc.:

>= 3.81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

other: fry survival

Details on results:

A. MORTALITY:
There were no statistically significant differences or dose-response effects in embryo viability, time-to-hatch, and mortality of juveniles between the spirodiclofen-treated levels and the controls. The EC50, NOEC, and LOEC for these endpoints are >3.81, 3.81, and >3.81 µg/L, respectively.

B. SUB-LETHAL TOXICITAYN D OTHER CHRONIC EFFECTS:
Egg hatching began on day 33 and continued until day 37 when it reached 100% in all test levels. There was no difference no difference in time to hatch in any treatment group compared to the pooled control data.
Swim-up of newly hatched fry began on study day 48 (post-hatch day 11). The swimming-up transient stage was observed between study days 48 and 60 (Table 7). Greater than 97 percent swim-up was achieved on day 55 in the controls. There was no difference in time to swim-up in any test treatment compared to the pooled control data. Growth was measured after study termination on study day 97 (post-hatch day 61). Analysis of length data showed slight reductions at the test levels 1.09, 1.95, and 3.81 µg/L in comparison to the pooled controls. Reduction in length was 5% or less up to 1.95 µg/L which is in the same range as the variation within the control replicates (pooled control mean: 33.4 mm; minimum: 31.8 mm; maximum: 35.4 mm). In the highest level (3.81 µg/L) the reduction was 7% and exceeded the range of control variability. Analysis of dry weight showed no statistical difference in dry weight in any treatment group compared to the pooled controls. Length and weight are considered as sensitive integrative endpoints for fry growth. Small (<= 5% relative to the pooled controls) reductions in length at concentrations up to 1.95 µg/L are not considered biologically significant, as they are in the range of the control variability and there was no significant reduction in weight simultaneously. The slight effect at 3.81 µg/L of 7% length reduction (statistically significant and exceeding control variability) together with 11% reduction in mean weight is considered biologically significant, though the weight reduction is not statistically significant.
There were no treatment-related morphological or behavioral effects observed during the study. The NOEC and LOEC for these endpoints are 1.95 µg/L and 3.81 µg/L, respectively, based on the growth parameters of length and weight.

Reported statistics and error estimates:

Replicate data for each concentration were grouped for analysis. Replicate means were used for
statistical analysis since each test chamber (aquarium) was the experimental unit based on the
design of the test system. For each parameter analyzed the following statistical tests were
conducted:
• t-test to determine if control and solvent control data can be pooled;
• chi-square test to test the normality of the data set; and
• Bartlett‘s test for homogeneity of variances.
Data which did not fit a normal distribution were analyzed by the non-parametric Kruskal-
Wallis’ test and Dunn’s Multiple Comparison test to determine if there was a significant
difference between the treatment and control groups.
Control data were pooled if the t-test criteria were met. If the data was not poolable only the
corresponding solvent control data was used for further analyses.
Fry survival, egg hatch, time to hatch and time to percent swim-up data were arcsine transformed
and analyzed using the Dunnett’s one-tailed multiple means comparison test, the Bonferroni-ttest,
the Tukey method of multiple comparisons and the Williams test (isotonic regression
model).
Growth data, expressed as dry weight and as length were analyzed using the Dunnett’s one-tailed
multiple means comparison test, the Bonferroni-t-test, the Tukey method of multiple
comparisons and the Williams test (isotonic regression model) without previous data
transformation.
All statistical analyses were conducted using a PC based computer program developed by
WEST, Inc. & D.G. Gulley with conclusions of statistical significance based on a 95
percent confidence level (a = 0.05).

Table 3: Effect of spirodiclofen technical on egg hatching and survival at different life stage of rainbow trout.

	Egg hatched/embryo viability
		hatch/embryo viability	‘’	Time to hatch (% hatch)	-	-	Juvenile-survival on day 97
Treatment (μg/L) [record measured and nominal concentrations used	No. of eggs at study initiation	No.	%*	Day 33	Day 35	Day 39	No. dead	% mortality
Control (dilution water only)	140	124	89(92)	1	66	96	7	10
Solvent control	140	128	91(95)	1	66	91	2	3
Pooled control	---	---	90(93)	1	66	90	---	7
0.112(0.123)	140	126	90(93)	1	57	90	1	2
0.29(0.25)	140	121	86(90)	0	81	86	5	8
0.57(0.49)	140	127	91(94)	0	74	91	1	2
1.09(0.98)	140	118	84(87)	0	69	84	3	5
1.95(1.97)	140	124	89(92)	1	77	89	1	2
3.81(3.93)	140	130	93(96)	0	60	93	3	5
NOEC	3.81μg/L	‘’	‘’	‘’	‘’	‘’	‘’	‘’
EC50	>3.81μg/L	‘’	‘’	‘’	‘’	‘’	‘’	‘’
Positive control, if used Mortality: EC50:  NOEC	NA	NA	NA	NA	NA	NA	NA	NA

*Egg hatch percent in parentheses are corrected for a hatch success factor of 0.965

 

 

Table 4: Effect of spirodiclofen technical on growth of juvenile rainbow trout

Treatment (μg/L) [record measured and nominal concentrations used	Swim-up (% swim-up)							)
	day 48	Day 50	Day 52	Day 55	Day 59	Day 60	Growth – length (mm)	Growth dry weight (g)
Control (dilution water only)	2	9	31	96	100	100	33.8	73.2
Solvent control	0	7	29	98	100	100	32.9	71.0
Pooled control	1	8	30	97	100	100	33.4	72.1
0.112(0.123)	2	15	40	100	100	100	33.0	69.5
0.29(0.25)	0	20	36	98	100	100	33.1	78.5
0.57(0.49)	2	10	41	100	100	100	32.7	69.5
1.09(0.98)	0	0	17	93	100	100	31.8*	70.2
1.95(1.97)	5	8	28	97	98	100	31.9*	71.4
3.81(3.93)	2	5	43	100	100	100	31.1*	64.1
NOEC	3.81 μg/L	‘’	‘’	‘’	‘’	‘’	1.95 μg/L	3.81 μg/L
EC50	>3.81 μg/L	‘’	‘’	‘’	‘’	‘’	>3.81 μg/L	>3.81 μg/L
Positive control, if used Mortality: EC50: NOEC	NA				NA	NA	NA	NA

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

The EC50, NOEC, and LOEC for this study are >3.81µg/L, 1.95µg/L, 3.81µg/L, respectively,
based on the growth (length and weight) as the most sensitive endpoint. This is a scientifically
sound study and should be classified as acceptable.

Executive summary:

The 97-day chronic toxicity of spirodiclofen technical to early life stage of rainbow trout (Oncorhynchus mykiss) was studied under flow-through conditions. Fertilized eggs (35 eggs per replicate for 140 eggs per test level; eggs were < 24 hours old at start of exposure) of rainbow trout were exposed to control, solvent control (acidified methanol), and nominal (measured) test chemical concentrations of 0.123 (0.112), 0.25 (0.29), 0.49 (57), 0.98 (1.09), 1.97 (1.95), and 3.93 (3.81) μg/L. The test system was maintained at 9.8 to 11.0°C and a pH of 6.4 to 7.3. The 97-day EC50 and NOEC values, based on mortality/sublethal effects (length/weight), were >3.81 μg/L and 1.95 μg/L, respectively. The parameters measured in this study were egg hatchability, survival and fry growth, expressed by different toxic endpoints (egg hatch, time to hatch, time to swim-up, fry survival, fry growth (length and weight), morphological and behavioral effects). No treatment-related or statistically significant effects were noted for any parameter except growth (length and weight). The most sensitive endpoint was growth. A statistically and biologically significant reduction in length and weight was seen at the highest level tested (3.81 μg/L).
This toxicity study is classified as acceptable and satisfies the guideline requirement for early life toxicity study with fish.
Results Synopsis Test Organism Size/Age(mean wet weight or length): <24-hour embryos used to initiate study
Test Type (Flowthrough, Static, Static Renewal): Flow-through
LOEC: 3.81 μg/L
NOEC: 1.95 μg/L
Endpoint(s) Effected: Growth (length and weight)

Description of key information

Oncorhynchus mykiss was studied under flow-through conditions. Fertilized eggs (35 eggs per replicate for 140 eggs per test level; eggs were < 24 hours old at start of exposure) of rainbow trout were exposed to control, solvent control (acidified methanol), and nominal (mean measured) test chemical concentrations of 0.123 (0.112), 0.25 (0.29), 0.49 (57), 0.98 (1.09), 1.97 (1.95), and 3.93 (3.81) μg/L. The test system was maintained at 9.8 to 11.0°C and a pH of 6.4 to 7.3. The 97-day EC50 and NOEC values, based on mortality/sublethal effects (length/weight), were >3.81 μg/L and 1.95 μg/L, respectively. The parameters measured in this study were egg hatchability, survival and fry growth, expressed by different toxic endpoints (egg hatch, time to hatch, time to swim-up, fry survival, fry growth (length and weight), morphological and behavioral effects). No treatment-related or statistically significant effects were noted for any parameter except growth (length and weight). The most sensitive endpoint was growth. A statistically and biologically significant reduction in length and weight was seen at the highest level tested (3.81 μg/L).
This toxicity study is classified as acceptable and satisfies the guideline requirement for early life toxicity study with fish.
Results Synopsis Test Organism Size/Age(mean wet weight or length): <24-hour embryos used to initiate study
Test Type (Flowthrough, Static, Static Renewal): Flow-through
LOEC: 3.81 μg/L
NOEC: 1.95 μg/L
Endpoint(s) Effected: Growth (length and weight)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

1.95 µg/L