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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

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 study is classified as acceptable and satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The 21-day-chronic toxicity of spirodiclofen technical to the waterflea, Daphnia magna, was studied under flow through conditions. Daphnids were exposed to the nominal concentrations of control, solvent control, 4.6, 8.3, 15.1, 27.5, and 50 µg/L. The mean measured concentrations were 4.39, 6.65, 11.1, 20.2, and 32.7 µg/L. Survival, growth as length and dry weight, behavior and sublethal effects, time to first brood, number of young produced per adult reproduction day.

Vehicle:

yes

Details on test solutions:

- Method: Test conditions: Flow-through using a Mount-Brungs diluter system with syringe pump
injection of solvent and toxicant. The flow rate provided 6.7 turnovers per day. Calibrated prior to study initiation. The diluter system and syringe pump were checked twice daily during the week and once on weekends. The diluter system operation was mechanically monitored 24 hours per day
- Controls: Four replicates at each level: control, solvent control, 4.6, 8.3, 15.1, 27.5 and 50 µg/L.
- Dilution water - Spring water blended with reverse osmosis water
- Solvent: acidified methanol (1% 0.1N HCl by volume) Solvent load was 0.1 mL/L

Test organisms (species):

Daphnia magna

Details on test organisms:

- Species: Daphnia magna
- Age of the parental stock: 16-day old adults produced first instar neonates
- Source: in house culture, original source was Aquatic Biosystems, Fort Collins, Colorado
USA.
- Parental subculture was 16 days old. The subculture conditions were very similar to test conditions, static-renewal in the same water as used for testing, 20 ± 2°C, 16 hours light; 8 hours dark, fed with
green algae (S. capricornutum and A. falcatus), light intensity of 50 footcandles. No specifics given on the health of the subculture.

Test type:

flow-through

Water media type:

freshwater

Total exposure duration:

21 d

Hardness:

Hardness: 160 to 164 mg/L as CaCO3

Test temperature:

Temp: 19.7 to 21.3°C.

pH:

pH: 8.0 - 8.2

Dissolved oxygen:

DO: 79 to 99% saturation

Nominal and measured concentrations:

Nominal concentrations: 4.6, 8.3, 15.1, 27.5 and 50 µg/L
Mean measured concentrations: 4.39, 6.65, 11.1, 20.2, and 32.7 µg/L

Details on test conditions:

Test System:
- Test vessel: Test chambers were borosilicate, 1-L glass beakers.
The test solution volume was 900 mL at a depth of approx. 11 cm. All test chambers were of the same size, volume and material.
- Aeration: aeration is not mentioned in the report.
- No. of organisms per vessel:10 organisms per replicate chamber; 4 replicates per treatment; 40 organisms per treatment level. All replicates were used to statistically analyze growth, reproduction and survival.
- No. of vessels per concentration (replicates): Four replicates at each level: control, solvent control, 4.6, 8.3, 15.1, 27.5 and 50 µg/L
- Biomass loading rate: Loading was 10 adult daphnids per 900 mL of test solution.

Water Parameters:
- Source/preparation of dilution water: Spring water blended with reverse osmosis water
- Total organic carbon: TOC: less than 1 mg/L
- Particulate matter: Total suspended solids: 2 mg/L
- Intervals of water quality measurement: Water quality measured on Day 0, once per week and on Day 21.
- Light intensity: 16 hours light and 8 hours dark. The light phase included a 30-minute transition period of lower intensity light at the initiation and termination of the light phase. Light intensity was 58 foot candles.

- Range finding study: Range-finding Study: No range find conducted. Definitive test levels were based upon a previous chronic test (Heimbach, 1998) and the water solubility limits in a flow-through diluter system (30 µg/L).

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 32.7 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

and time to first brood

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

20.2 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

young/adult/reproduction day

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

32.7 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

Adult body length and adult dry weight

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

32.7 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

and time to first brood

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

11.1 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

young/adult/reproduction day

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

20.2 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

Adult body length and adult dry weight

Details on results:

A. MORTALITY:
Survivorship ranged from 60% to 100% across individual test vessels, however mean percent survivorship by treatment ranged from 88 to 100%. The control replicate A test vessel had the lowest survivorship (60%), however two other control replicates had 100% survivorship and 100% survivorship was noted in some replicates of all test levels. The no observed effect concentration (NOEC) for the survival endpoint was 32.7 µg/L and the 21-day EC50 was greater than 32.7 µg/L. The control daphnids produced adequate numbers of neonates over the 21 day exposure. On average, the control and solvent control daphnids produced from 67 to 115 neonates per adult.

B. EFFECT ON REPRODUCTION AND CHRONIC/SUBLETHAL EFFECTS :
Analytical results:
The mean measured concentrations of spirodiclofen for the study were 4.39, 6.63, 11.1, 20.2, 32.7 µg/L for the nominal test levels of 4.6, 8.3, 15.1, 27.5, 50.0 µg/L, respectively. Analytical recovery was the lowest in the highest test level, which reflects the practical limit of solubility (approximately 30 µg/L) (Kendal and Nixon, 2000). The highest test level was chosen to replicate the study performed by Heimbach (1998). No undissolved test substance was observed in the test chambers. The 27.5 µg/L test level had one analytical measurement that appeared to be high. Additional analyses with that test level were conducted on Day 1 to confirm the concentration of spirodiclofen in that replicate. All subsequent observations and discussions will refer to mean measured concentrations of the test solutions.

Sublethal effects:
For the Daphnia with which the test was initiated (referred to as the adults), one daphnid on bottom was noted in the control, 11.1 and 20.2 µg/L test levels. One pale organism was noted in the solvent control, 6.63 and 32.7 µg/L test levels. On Day 21, one replicate of the 32.7 µg/L test level had 10 pale organisms. These findings were not considered to be biologically relevant since the observations were also noted in the control or solvent control and/or the observation was only noted a single replicate. For the neonates produced during the test in the beakers used to assess reproduction, no sublethal effects were observed in the control or solvent control. A single unhatched egg out of the hundreds of neonates produced was observed in the 4.39 and 11.1 µg/L test levels. A total of ten to thirteen unhatched eggs, out of the hundreds of neonates produced, were noted in the test levels greater than or equal to 6.63, 20.2 and 32.7 µg/L test levels. A few neonate mortalities were observed (one to seventeen), in the 11.1, 20.2, and 32.7 µg/L test levels. These findings were not considered to be biologically relevant.

Time to first brood:
The mean time to first brood ranged from 8.0 to 10.0 days. A significant difference between the control and solvent control were noted for the time to first brood (P> 0.05), therefore the solvent control group was used for statistical analyses. No significant differences between the solvent control and spirodiclofen test levels were detected for the time to first brood, resulting in a NOEC of 32.7 µg/L.

Neonates per adult reproduction day:
The mean number of neonates per reproductive day ranged from 2.78 to 6.15. The control replicate A was a clear outlier for this parameter and therefore was not included in the statistical analysis. As mentioned earlier, this control replicate had the highest mortality rate, which may have influenced neonate production via a greater abundance of food availability. Given that the other control and solvent control replicates had good biological responses, the impact of excluding this control replicate from the number of neonates per reproduction day endpoint should have minimal impact on the validity of the study. No significant difference between the control and solvent control were detected (p <0.05), therefore the two control groups were pooled for the remaining data analysis. Statistical analysis (ANOVA followed by Dunnett’s test) of the young (neonates) per adult reproduction day for Daphnia magna during the 21-day exposure to spirodiclofen indicated that only the 20.2 and 32.7 µ­g/L test levels had significantly reduced reproduction compared to the pooled control. The no-observed-effect-concentration (NOEC) was 11.1 µg/L.

Growth:
No significant difference was detected between the control and solvent control, therefore the pooled control was used for the remaining statistical analyses. The ANOVA followed by Dunnett’s test demonstrated that the 32.7 µg/L test level was significantly different from the pooled control. The NOEC for dry weight was 20.2 µg/L. No significant difference was detected between the control and solvent control, therefore the pooled control was used for the remaining statistical analyses. The ANOVA followed by Dunnett’s test demonstrated that the 32.7 µg/L test level was significantly different from the pooled control. The NOEC for length was 20.2 µg/L.

Reported statistics and error estimates:

Reproductive data consisted of two parameters indicative of reproductive success: time to first
brood and the number of offspring produced per adult reproduction day. The data from each test
chamber was used for the reproduction data statistical analysis since the experimental unit for
this study was the replicate test chambers. The number of offspring produced per adult
reproduction day for each parent daphnid was calculated by: Total Number of Offspring Produced in 21 Days Number of Reproduction Days The number of reproduction days was equal to the number of days from the onset of neonate production to test termination times the number of adult daphnids present on each day. The number of reproductive days accounts for mortalities that may have occurred in any one replicate. For length and weight parameters, the experimental unit is equal to replicate test chambers, and hence the statistical analysis was performed with the n equal to 4 for the four replicate chambers per treatment group. For each analyzed parameter the following statistical tests were conducted: 1) chi-square test to test the normality of the data set; 2) Bartlett’s test for homogeneity of variances. A T-test was used to determine if the control and solvent control were statistically different. If no statistical difference was detected then the two control groups were pooled. However if statistical difference was detected between the control and solvent control, then the solvent control group was used in the subsequent analyses. To determine if the spirodiclofen treatment groups were significantly different from the control, the reproduction data, survival data and parent daphnid growth data (length and dry weight) were analyzed by ANOVA followed by the Dunnett’s test (if the control groups were not pooled), the Bonferroni t-test (if the control and solvent control groups were pooled), or a nonparametric analysis if the assumptions of normality and/or homogeneity of variance were not met.

Table 3: Effect of spirodiclofen technical on growth and survival of Daphnia magna

Treatment (µg/L) [measured conc.]	Mortality (dead or immobile)		Length (mm)	Dry weight (mg)
	No. dead	%
Control	5	12	4.31	0.667
Solvent control	3	10	4.34	0.681
4.39	0	0	4.35	0.713
6.65	4	10	4.36	0.763
11.1	2	5	4.26	0.651
20.2	4	10	4.23	0.624
32.7	1	2	3.71	0.308
NOEC	32.7		20.2	20.2
LOEC	>32.7		32.7	32.7

Validity criteria fulfilled:

yes

Conclusions:

Study is acceptable.
The most sensitive end point was neonate per adult reproduction day.
LOEC 20.2 µg/L
NOEC 11.1 µg/L

Executive summary:

The 21-day-chronic toxicity of spirodiclofen technical to the waterflea, Daphnia magna, was studied under flow through conditions. Daphnids were exposed to the nominal concentrations of control, solvent control, 4.6, 8.3, 15.1, 27.5, and 50 µg/L. The mean measured concentrations were 4.39, 6.65, 11.1, 20.2, and 32.7 µg/L. The 21-day LC50 was greater than the highest test concentration of 32.7 µg/L. The 21-day NOEC based on the reproductive endpoint of young per adult reproduction day was 11.1 µg/L. The sublethal effects noted during the 21-day exposure included pale color, abnormal position at the bottom of the test vessel, and unhatched neonates. There was no dose-response relationship for any of these sublethal effects, therefore were not compound related. The number of organisms noted to have sublethal effects was very minimal and none of these sublethal affects were biologically significant. Production of offsprings in the treated groups indicated that spirodiclofen technical had an effect on the reproduction at 20.2 and 32.7 µg/L. The most sensitive end point was neonate production as young per adult reproduction day. This study is classified as acceptable and satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates.
Results Synopsis
Test Organism Age: first instar, less than 24 hours old at initiation
Test Type: Flow through
NOEL: 11.1 µg/L
LOEC: 20.2 µg/L Endpoint(s) Effected: young per adult reproduction day

Description of key information

The 21-day-chronic toxicity of spirodiclofen technical to the waterflea, Daphnia magna, was studied under flow through conditions. Daphnids were exposed to the nominal concentrations of 4.6, 8.3, 15.1, 27.5, and 50 µg/L, a control and solvent control. The mean measured concentrations were 4.39, 6.65, 11.1, 20.2, and 32.7 µg/L. The 21-day LC50 was greater than the highest test concentration of 32.7 µg/L. The 21-day NOEC based on the reproductive endpoint of young per adult reproduction day was 11.1 µg/L. The sublethal effects noted during the 21-day exposure included pale color, abnormal position at the bottom of the test vessel, and unhatched neonates. There was no dose-response relationship for any of these sublethal effects, therefore they were not compound related. The number of organisms noted to have sublethal effects was very minimal and none of these sublethal affects were biologically significant. Production of offsprings in the treated groups indicated that spirodiclofen technical had an effect on the reproduction at 20.2 and 32.7 µg/L. The most sensitive endpoint was neonate production as young per adult reproduction day. This study is classified as acceptable and satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates.
Results Synopsis
Test Organism Age: first instar, less than 24 hours old at initiation
Test Type: Flow through
NOEL: 11.1 µg/L
LOEC: 20.2 µg/L Endpoint(s) Effected: young per adult reproduction day

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

11.1 µg/L