thiacloprid (ISO);(Z)-3-(6-chloro-3-pyridylmethyl)-1,3-thiazolidin-2-ylidenecyanamide;{(2Z)-3-[(6-chloropyridin-3-yl)methyl]-1,3-thiazolidin-2-ylidene}cyanamide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 - 24 Apr 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 202, Part II (Reproduction Test)

Version / remarks:

1984

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 0.10, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6 and 10 mg a.i./L
- Sampling method: Samples were taken prior to introducing the food and test animalson days 0, 4, 11 and 18. At day 7, 14 and 21 a test for the stability of the test substance was performed with all test concentrations. For each of the concentrations one container without algae (test solution before introducing of food) was exposed to the test conditions. After 72 hours samples were taken. Samples with algae were taken from the test solutions of the ongoing study.
- Sample storage conditions before analysis:

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test concentrations were set up new at all transfer times as follows: 71.9 mg of the test item were dissolved in 1000 mL of warm (about 35 °C) test water, ultrasonicated for 5 minutes and then made up to 7 L with cold (about 20 °C) test water (= stock solution). Of this stock solution 30 mL, 96 mL, 168 mL, 300 mL, 540 mL, 960 mL and 1680 mL were each made up to 3000 mL with test water in order to produce the test concentrations.
- Differential loading: no
- Controls: dilution water only
- Test concentration separation factor: 3.2

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: genotype No. 2 (type B)
- Age at study initiation: < 24 hours
- Method of breeding: kept in an environmental chamber under the test conditions of 20 ± 1 °C, 16 : 8 hour light-dark cycle; the animals are fed with single cell green algae Scenedesmus subspicatus and occasionally some commercial ornamental fish food (trade name: TetraMin®) (aqueous suspension)
- Source: laboratory breeding stock originally obtained from Bundesgesundheitsamt in Berlin, Germany 10 years ago
- Age of parental stock (mean and range, SD):
- Feeding during test
- Food type: living single cell green algae (Scenedesmus subspicatus)
- Amount: 2 x 10^8 cells/L from Mondays - Thursdays, 2 x 10^8 cells/L on Fridays
- Frequency: daily during the week, but not at weekends

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: first instars were obtained by repeated carefully screening of adults (14-21 days old, only parents of the same age (± 12 hours) were used) using plastic screens with 0.6 and 0.2 mm mesh

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

12 mg CaCO3/L

Test temperature:

20.0 - 20.4 °C

pH:

7.63 - 8.53

Dissolved oxygen:

7.9 - 10.2 mg/L

Conductivity:

580 - 610 µS/cm

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.10, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6 and 10 mg a.i./L
Mean measured concentrations: < LOD (control), 0.11, 0.33, 0.58, 1.05, 1.85, 3.30, 5.80, 10.0 mg a.i./L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL beakers
- Type: closed
- Material, headspace, fill volume: glass, 50 mL, 200 mL
- Aeration: no
- Renewal rate of test solution: every 48 hours during the week, and at 72 hours over weekends
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water: M7-medium according to OECD guideline 211
- Culture medium different from test medium: no
- Intervals of water quality measurement: oxygen and pH ( in freshly prepared test solutions of the control, and all test concentrations), total hardness and alkalinity (weekly in controls and all test concentrations)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16:8 h light:dark
- Light intensity:

EFFECT PARAMETERS MEASURED: reproduction (3 times per week), body length and weight (on day 21)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

body length

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

- Observations on body length and weight: statistically significant differences in body length at the all test concentrations higher than 0.58 mg a.i./L, statistically significant differences in body weightat the three highest test concentrations (3.30, 5.80 and 10 mg a.i./L)
- Other biological observations:
- Mortality of control: 0%

Results with reference substance (positive control):

Relevant effect levels: EC50 (24 h) = 1.46 mg/L

Table 1: Summary of analytical results

Time (d)	Nominal concentrations (mg a.i./L)
	Control	0.10	0.32	0.56	1.0	1.8	3.2	5.6	10
0 (new)	< LOD	0.113	0.338	0.600	1.06	1.89	3.36	5.78	10.1
4 (new)	< LOD	0.108	0.330	0.590	1.05	1.88	3.33	5.91	10.0
7 (old)	< LOD	0.112	0.338	0.597	1.07	1.89	3.35	5.91	10.1
11 (new)	< LOD	0.106	0.334	0.595	1.07	1.83	3.34	5.90	10.0
14 (old)	< LOD	0.104	0.340	0.624	1.08	1.88	3.49	6.00	10.1
18 (new)	< LOD	0.106	0.314	0.554	1.01	1.79	3.17	5.61	9.97
21 (old)	< LOD	0.094	0.325	0.569	1.02	1.84	3.24	5.82	10.2

In order to ascertain the stability of the active ingredient during the study, a parallel study without food was performed at all concentrations; the active ingredient was analyzed on days 4, 11 and 18 each time after 72 hours exposure of the test medium under the test conditions. The results of the samples without food show that the measured concentrations at the end of 72 hours exposure was not lower than at the start of the study. In order to specify the amount of active ingredient dissolved in the water or possibly adsorbed onto the algal cells and other particles in the test medium of the daphnia study, the suspended particles were centrifuged from the original 3 day-old food containing test solutions, prior to analysis and the supernatant (clear water) was analyzed. The results show that the test substance was not adsorbed to algae or other particles.

Table 2: Survival of parental water fleas after 21 days

Mean measured concentration [mg a.i./L]	Survival (%)
Control	100
0.11	100
0.33	100
0.58	93
1.05	100
1.85	100
3.30	100
5.80	100
10.0	100

Table 3: Mean reproduction after 21 days

Mean measured concentration [mg a.i./L]	Number of newborn daphnids/replicate
Control	123.3
0.11	130.4
0.33	141.5
0.58	145.2
1.05	136.9
1.85	114.1
3.30	106.0
5.80	91.5*
10.0	45.1*

*statistical significance to control

Table 4: Mean body length after 21 days

Mean measured concentration [mg a.i./L]	Body length (mm)
Control	4.54
0.11	4.50
0.33	4.44
0.58	4.42
1.05	4.33*
1.85	4.02*
3.30	3.80*
5.80	3.60*
10.0	3.03*

*statistical significance to control

Table 5: Validity criteria for OECD 211

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals in the control (female Daphnia) does not exceed 20% at the end of the test.	0%	yes
The mean number of living offspring produced per parent animalsurvivingat the end of the test is ≥ 60 in the control.	123.3	yes

Validity criteria fulfilled:

yes

Remarks:

For further, details, please refer to 'Any other information on results incl. tables'.

Conclusions:

The lowest derived NOEC (21 d) in the Daphnia magna reproduction test was determined to be 0.58 mg a.i./L (mean measured) based on body length.

Reference 2

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 0.25, 0.50, 1.0, 2.0, 4.0 and 8.0 µ a.i./L for liquid scintillation counting (LSC) and 8 µg/L for HPLC analysis
- Sampling method: collected from alternating replicate test chambers from mid-depth of each test chamber on day 0, 7, 14, 21, 28 and 32 (LSC)
- Sample storage conditions before analysis: Samples were analyzed as soon as possible after collection without storage.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: radiolabelled test item stock solution of 100 g/L and stock solution of non-labelled target substance of 80 mg/L were prepared and used for further dilution to prepare secondary stock solutions. All working stocks were brought to a final volume of 500 mL with RO water. The nominal radioactivity in each working stock was 357,143 dpm/mL. Stocks were prepared a second time on Day 21 of the test. The procedures followed were similar to those above except that the final volume of each stock was 250 mL. The stock solutions were diluted with filtered saltwater in the mixing chambers in order to obtain the desired test concentrations.
- Differential loading: no
- Controls: dilution water only
- Test concentration separation factor: 2
- Evidence of undissolved material: not reported

Test organisms (species):

other: Mysidopsis bahia

Details on test organisms:

TEST ORGANISM
- Common name: saltwater mysid
- Age at study initiation: < 24 h
- Source: obtained from cultures maintained by the testing facility
- Feeding during test : yes
- Food type: live brine shrimp (Artemia sp.) nauplii
- Amount: not reported
- Frequency: at least 2 times a day

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: Juvenile mysids were carefully collected one or two at a time from the
cultures held in water from the same source as used during the test.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

32 d

Test temperature:

26.2- 27.2 °C

pH:

8.1 - 8.2

Dissolved oxygen:

≥ 5.1 mg/L (72% of saturation at 27°C and 20‰)

Salinity:

20‰

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.25, 0.50, 1.0, 2.0, 4.0 and 8.0 µg a.i./L
Mean measured concentrations: < LOQ (control), 0.28, 0.54, 1.1, 2.2, 4.4 and 8.5 µg a.i./L

Details on test conditions:

TEST SYSTEM
- Test vessel: 12 cm diameter dishes (prior sexual maturity), 5 cm petri dishes (reproductive compartment)
- Type: closed (nylon mesh screen attached with silicone adhesive)
- Test apparatus: The test compartments were placed in 18.9-L glass aquaria. A glass vial was inverted over the standpipe and this provided automatic cycling of the water depth between 5.5 and 9 L. The depth in the test chambers fluctuated between 6.3 and 10 cm.
- Aeration: no
- Type of flow-through: continuous-flow diluter
- Renewal rate of test solution: 14 volume additions/day
- No. of organisms per vessel: 10 (prior sexual maturity), 10 male/female pairs (reproductive compartment)
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: natural seawater obtained at Indian River Inlet, Delaware, USA and diluted to a salinity of approximately 20%o with well water. Freshly-collected seawater was passed through a sand filter (25 µm) and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to delivery to the diluter system, the water again was filtered to remove microorganisms and particles.
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature (test start and end and at wekly intervals), sex and maturity of each mysid (time of pairing),

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16:8 h, light:dark
- Light intensity: 359 lux

EFFECT PARAMETERS MEASURED: mortality and clinical signs of toxicity in first-generation mysids (daily), number of second-generation mysids (daily), abnormal development and aberrant behavior, sex of each surviving first-generation mysid, dry weight and length of each mysid (test end)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Reference substance (positive control):

no

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

0.608 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: Statistical recalculation in M-581576-01-1

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

1.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

length and dry weight

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: Mean length and dry weight were significantly reduced in the 2.2, 4.4 and 8.5 µg/L treatment groups.
- Other biological observations: Reproduction was significantly reduced in the 2.2, 4.4 and 8.5 µg/L treatment groups.
- Mortality of control: 14%

Reported statistics and error estimates:

Survival data were analyzed using 2 x 2 contingency tables and the Chisquare test to identify treatment groups that were statistically different from the control group (p≥0.05). The analyses of reproduction (number of live young produced per reproductive day) and growth (dry weights and lengths) data included all treatments which did not exhibit a statistical reduction in survival. Analyses included the evaluation of homogeneity of variances using Bartlett's test and the assessment of normality using a goodness of fit test (e.g., Chi-square test or equivalent). When data were deemed normal and homogeneous, an analyses of variance test was used to determine whether or not statistically significant differences existed among experimental groups. Those treatments statistically different from the control group were identified using the Bonferonni t-test (p≤0.05). When data failed the tests for normality and homogeneity and no data transformations corrected the condition, the Kruskal-Wallis Test was used to evaluate treatment-related effects.

Table 1: Analytical results

 

Nominal test concentration (µg a.i./L)	Replicate	Measured concentration (µg a.i./L)
		Day 0	Day 7	Day 14	Day 21	Day 28	Day 32	Mean measured concentration (µg a.i./L)	Percent of nominal
Control	A	-	< LOQ	-	< LOQ	-	< LOQ	< LOQ	-
	B	< LOQ^1	-	< LOQ	-	< LOQ	-
0.25	A	-	0.28	-	0.30	-	0.28	0.28	112
	B	0.28	-	0.29	-	0.25	-
0.50	A	-	0.54	-	0.58	-	0.55	0.54	108
	B	0.50	-	0.56	-	0.48	-
1.0	A	-	1.0	-	1.1	-	1.0	1.1	110
	B	1.1	-	1.1	-	1.0	-
2.0	A	-	2.2	-	2.3	-	2.2	2.2	110
	B	2.1	-	2.2	-	2.1	-
4.0	A	-	4.6	-	4.2	-	4.2	4.4	110
	B	4.3	-	5.1	-	4.0	-
8.0	A	-	9.3^2	-	8.7^2	-	8.6^2	8.5	106
	B	7.9^2	-	8.4^2	-	7.9^2	-

¹: LOQ = 0.023 µg/L based on twice average background measured during the test (36.44 dpm)

²: 75 – 91% of the nominal concentration was confirmed to be parent compound based on HPLC.

Table 2: Effects of the target substance on mysid mortality, reproduction and growth

Nominal test concentration (µg a.i./L)
	Percent mortality	Mean number of young/female	Total Length (mm) Mean (±SD)		Dry Weight (mg) Mean (±SD)
Control	14%	3.84	6.78 (±0.44)		0.65 (±0.12)
0.25	13%	3.50	6.76 (±0.38)		0.68 (±0.11)
0.50	13%	2.80	6.73 (±0.39)		0.67 (±0.1 2)
1.0	17%	3.00	6.69 (±0.40)		0.64 (±0.12)
2.0	18%	1.06	6.38 (±0.32)*		0.58 (±0.10)*
4.0			6.21 (±0.34)*		0.56 (±0.11)*
8.0	33%	0	5.92 (±0.31)*		0.50 (±0.098)*

*Indicates a significant diffenrence from the control

Table 1: Validity criteria for OPPTS 850.1350 Mysid Chronic Toxicity Test

Criterion from the guideline	Outcome	Validity criterion fulfilled
At least 70% survival of control animals in all test phases over the duration of the chemical exposure	86%	yes
At least 75% of the females produce young	At the time of pairing, the sex and maturity of each mysid was determined by microscopic examination and immature mysids were discarded.	yes
The average number of young produced per female in the controls is > 3 after 28 days.	3.84 young/female after 32 days	yes

Validity criteria fulfilled:

yes

Remarks:

For further, details, please refer to 'Any other information on results incl. tables'.

Conclusions:

The NOEC (32 d) in a life-cycle toxicity test with Mysidopsis bahia was 1.1 µg a.i./L (mean measured). The EC10 (32 d) was statitstically reevaluated and was determined to be 0.608 µg a.i./L (mean measured).

Description of key information

NOEC (21 d) = 0.58 mg a.i./L (mean measured, OECD 202, part II, Daphnia magna)

EC10 (32 d) = 0.608 µg a.i./L (mean measured, Americamysis bahia, EPA OPP 72-4)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.58 mg/L

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

EC10

Effect concentration:

0.608 µg/L

Additional information

In the key study, water flea Daphnia magna were exposed for 21 days to a nominal test item concentration range between 0.1 and 10 mg a.i./L in a semi-static test design according to OECD 202, part II. The mean measured concentrations were 0.11, 0.33, 0.58, 1.05, 1.85, 3.30, 5.80 and 10.0 mg a.i./L. Compared to the control, there was no statistically significant reduction of the sum of offspring per parent and the number of offspring per parent and reproduction day at the test concentrations of 0.11 to 3.30 mg a.i./L. In the concentrations 5.80 and 10.0 mg a.i./L the sum of offspring per parent and the number of offspring per parent and reproduction day was significantly reduced compared to the control. In the concentrations range from 1.05 to 10.0 mg a.i./L, the body length was significantly reduced compared to the control and the dray weight of parental daphnids was significantly decreased at 3.30 mg a.i./L and higher concentrations. The lowest derived NOEC (21 d) was therefore determined to be 0.58 mg a.i./L based on body length and mean measured concentrations.

 

The result of the freshwater key study is supported by a flow-through life-cycle toxicity saltwater key study with the saltwater mysid (Mysidopsis bahia). The test was performed under flow-through conditions according to FIFRA Guideline 72 -4. Neonates, less than 24 h old, were exposed to nominal test item concentrations between 0.25 and 8.0 µg a.i./L for 32 days. Mean measured concentrations ranged from 106 - 112 % of the nominal concentrations. The lowest derived effect value was an EC10 (32 d) of 0.608 µg a.i./L based on reproduction and mean measured concentrations.