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

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Jan 2013 - 17 Mar 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 233 (Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sediment)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Additional prepared test vessels were analysed and samples of the overlying water of cristalling dishes (oviposition phase) were analysed one time, one hour after spiking.

SEDIMENT
- Concentrations: 0 (control), 1.80 and 3.20 µg a.s./L
- Sampling interval: 1 hour, 7 days and 28 days after test item application

PORE WATER
- Concentrations: 0 (control), 0.32, 0.56, 1.00, 1.80 and 3.20 µg a.s./L
- Sampling interval: 1 hour, 7 days and 28 days after test item application

OVERLYING WATER
- Concentrations: 0 (control), 0.32, 0.56, 1.00, 1.80 and 3.20 µg a.s./L
- Sampling interval: 1 hour, 7 days and 28 days after test item application

Vehicle:

no

Details on sediment and application:

PREPARATION OF SPIKED WATER
- Details of spiking: Appropriate amounts of 38.7 (F1) mg/L stock solutions were applied into the overlying water column of the vessels as a singular event on study day 0, just below the water surface with a pipette by gently mixing of the water body ensuring homogeneous distribution of the test item without disturbance the sediment. For F2-generation exposure 38.7 - 38.8 mg/L stock solutions were used.
- Controls: dilution water only

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Common name: harlequin fly
- Justification for species other than prescribed by test guideline:
- Source: laboratory culture originally obtained from University of Frankfurt am Main, Germany in 2006
- Breeding conditions: Midges are kept in cages (40 x 40 x 40 cm), with a gauze on each side of the cage. Suitable basins are set on the bottom of each cage. The bottom of the basins are covered with a thin layer of "Kieselgur" (silica) and a 4 - 5 cm high layer of reconstituted water M7 according to Elendt, which is aerated gently. To start a culture in the cage, 2 - 4 egg masses are placed into the prepared basin. The hatched larvae are fed with green algae and an aqueous suspension of a plant material based fish food (Tetra Phyll®). After mating, female adults will lay egg masses on the water surface where these can be taken to start a new culture or to perform toxicity tests. The culture conditions are 20 ± 2°C and 16 to 8 hours light-darkcycle (light intensity ca. 500 - 1000 lux).
- Age of animals at beginning of exposure: First instar larvae L1
- Feeding during test
- Food type: commercial ornamental fish food extract (trade name Tetra Phyll®)
- Amount: 0.5 - 1 mg/larvae/day
- Frequency: at least about three times per week

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

larvae from first generation (P)

Duration:

28 d

Exposure phase:

larvae from second generation (F1)

Hardness:

284.8 - 320.4 mg CaCO3/L

Test temperature:

20.1 - 20.6 °C

pH:

8.3 - 8.9

Dissolved oxygen:

7.9 - 8.9 mg/L

Ammonia:

Ammoinum: 1.30 - 19.4 mg/L

Nominal and measured concentrations:

Nominal concentratiosn: 0 (control), 0.32, 0.56, 1.00, 1.80 and 3.20 µg a.i./L
Mean measured concentrations: Please refer to 'Any other information on results incl. tables'

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 0.6 L glass beakers (Ø 9.5 cm)
- Sediment volume: 140 g
- Weight of wet sediment with and without pore water:
- Overlying water volume: 0.38 L
- Depth of sediment and overlying water: 7.5 cm
- Aeration: yes
- Aeration frequency and intensity: approximately 2 bubbles per second

EXPOSURE REGIME
- No. of organisms per container (treatment): 20
- No. of replicates per treatment group: 8
- No. of replicates per control: 8
- Food type: commercial ornamental fish food extract (trade name Tetra Phyll®)
- Amount: 0.5 - 1 mg/larvae/day
- Frequency: at least about three times per

OVERLYING WATER CHARACTERISTCS
- Type of water: M7 medium acc. to OECD 219

CHARACTERIZATION OF ARTIFICIAL SEDIMENT
- % dry weight of sphagnum moss peat: 4
- Composition (if artificial substrate): 75.0 % quartz sand (grain size: 91.4% of the particles were in the range of 50-200 µm, originated form Quarzwerke GmbH D-50226 Frechen, Germany), 4% sphagnum moss peat (pH 3.5, particle size of ≤ 1 mm, air dried, originated from Erdenwerk Gregor Ziegler GmbH D-95703 Plößberg, Germany), 20 % kaolinite (kaolinite content of 30.2 %, pH value 5.5 – 7.5, "Kaolin W", from Erbslöh / Geisenheim, Germany), 0.1 % calcium carbonate (chemical pure, Merck) to adjust the pH value to 7 ± 0.5 and 48% deionised water.
- Method of preparation (if artificial substrate): Sediment was prepared 7 days before the start of the exposure period.

OTHER TEST CONDITIONS
- Photoperiod: 16 to 8 hours (light to dark)
- Light intensity: 500 - 1000 lux

EFFECT PARAMETERS MEASURED: behaviour (at least three times per week), sex, time point of emergence and number of emerged midges (daily), egg fertility, egg ropes: development and morphology of larvae (for at least six days after they have been produced)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 1.75
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (in August 2012)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.56 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

fertility

Remarks on result:

other: F1 generation

Details on results:

- Mortality of test animals at end of exposure period: Control mortality F1: 13.8%, control mortality F2: 3.8%
- Percent emergence per replicate and test concentration: F1 control: 86.3%, F2 control: 96.3%

Results with reference substance (positive control):

- Results with reference substance valid? yes (meets the range of 0.50 mg/L – 3.6 mg/L defined by OECD 235)
- Dose-response test: yes
- ECx: EC50 (48 h) = 2.62 mg/L

Table 1: Analytical results in F1 test setup

Nominal conc. [µg a.i./L]	Analytical results of the test item, means of two analyses each [µg a.i./L] / [µg a.i./kg sed.(dw)]
	1 hour / day 0		day 7		day 28
	analysedconc.	% of initial nominal per test vessel3)4)	analysedconc.	% of initial nominal per test vessel3)4)	analysedconc.	% of initial nominal per test vessel3)4)
control	Overlying water
	< LOQ1)	-	< LOQ1)	-	< LOQ1)	-
0.32	0.3222)	101	0.137	43	0.0548	17
0.56	0.4412)	79	0.203	36	0.070	13
1.00	0.7702)	77	0.317	32	0.125	13
1.80	1.542)	86	0.650	36	0.247	14
3.20	3.282)	103	1.42	44	0.517	16
average %		89		38		14
	Pore water
control	< LOQ1)	-	< LOQ1)	-	< LOQ1)	-
0.32	0.0342	0.7	0.065	1.5	0.0439	0.9
0.56	0.0935	0.9	0.0866	0.9	0.0457	0.5
1.00	0.0605	0.4	0.148	1.0	0.114	0.7
1.80	0.125	0.4	0.330	1.2	0.220	0.8
3.20	0.276	0.5	0.830	1.6	0.467	1.0
average %		0.6		1.2		0.8
	Sediment
control	< LOQ1)	-	< LOQ1)	-	< LOQ1)	-
0.32	n.a.	-	n.a.	-	n.a.	-
0.56	n.a.	-	n.a.	-	n.a.	-
1.00	n.a.	-	n.a.	-	n.a.	-
1.80	0.284	3.9	3.85	53	3.92	54
3.20	0.497	3.8	6.15	48	8.23	64
average %		3.9		51		59

1)    Foranalysisoftheoverlyingwaterandporewater, thelimitofquantitation(LOQ)ofthemethodis0.0311 µg thiacloprid /L, for the analysis of the sediment the limit of quantitation (LOQ) of the method is 0.05 µg thiacloprid /kg sediment(dw).

2)    Analytical results of thiacloprid, means of four analyseseach

3)  calculated to the real volume of pore water per test vessel

4)  calculatedtotherealamountofdryweightsedimentpertestvessel=93.94g(day-7).

n.a. = not analysed

Table 2: Analytical results in F2 test setup

Nominal conc. [µg a.i./L]	Analytical results of test substance, means of two analyses each [µg a.i./L] / [µg a.i./kg sed.(dw)]
	1 hour / day 0		day 7		day 28
	analysedconc.	% of initial nominal per test vessel3)4)	analysedconc.	% of initial nominal per test vessel3)4)	analysedconc.	% of initial nominal per test vessel3)4)
control	Overlying water
	< LOQ1)	-	< LOQ1)	-	< LOQ1)	-
0.32	0.300)	94	0.126	39	0.0348	11
0.56	0.485	87	0.188	34	0.0484	9
1.00	0.778	78	0.323	32	0.101	10
1.80	1.690	94	0.749	42	0.228	13
3.20	2.820	88	1.240	39	0.337	11
average %		88		37		11
	Pore water
control	< LOQ1)	-	< LOQ1)	-	< LOQ1)	-
0.32	< LOQ1)	0.0	0.0388	0.8	< LOQ1)	0.0
0.56	0.0389	0.5	0.0855	1.1	0.0416	0.5
1.00	0.0680	0.5	0.154	1.0	0.0677	0.5
1.80	0.1630	0.6	0.359	1.4	0.176	0.7
3.20	0.2180	0.5	0.516	1.2	0.235	0.5
average %		0.4		1.1		0.5
	Sediment
control	< LOQ1)	-	< LOQ1)	-	< LOQ1)	-
0.32	n.a.	-	n.a.	-	n.a.	-
0.56	n.a.	-	n.a.	-	n.a.	-
1.00	n.a.	-	n.a.	-	n.a.	-
1.80	0.316	4.4	3.13	43	3.50	48
3.20	0.241	1.9	4.74	37	5.17	40
average %		3.2		40		44

1) For analysis of the overlying water and pore water, the limit of quantitation (LOQ) of the method is 0.0311 µg thiacloprid /L, for the analysis of the sediment the limit of quantitation (LOQ) of the method is 0.05 µg thiacloprid /kg sediment (dw).

3) calculated to the real volume of pore water per test vessel.

4) calculated to the real amount of dry weight sediment per test vessel = 94.50 g (day 15).

n.a. = not analysed

Table 3: Influence on emergence and development rate after 29 days for F1-generation (based on nominal concentrations of the test item in the overlying water):

Nominal test concentration µg a.i./L	Number of emerged midges (introduced midges)	Emergence of inserted larvae (pooled sex)			Development rate^#)
					(1 / d)
		total (% )	male (% )	female (% )	pooled sex	male	female
control	138        (160)	86.25	47.50	38.75	0.064	0.066	0.062
0.32	140        (160)	87.50	46.25	41.25	0.063	0.064	0.061
0.56	142        (160)	88.75	37.50	51.25	0.062	0.065	0.060
1.00	137        (160)	85.63	44.38	41.25	0.063	0.065	0.061
1.80	141        (160)	88.13	48.75	39.38	0.062*	0.065	0.058*
3.20	74         (160)	46.25*	22.5	23.75	0.049*	0.051*	0.046*

* significant difference (α= 0.05)

#) for calculation of the true development time, the day of emergence was corrected to + 1 day, because the larvae had been introduced one day prior to application for the F1-generation, resulting in a study duration of 29 days.

Table 4: Influence on emergence and development rate after 28 days for F2-generation (based on nominal concentrations of the test item in the overlying water):

Nominal test concentration µg a.i./L	Number of emerged midges (introduced midges)	Emergence of inserted larvae (pooled sex)			Development rate
					(1 / d)
		total (% )	male (% )	female (% )	pooled sex	male	female
control	154        (160)	96.25	45.00	51.25	0.059	0.064	0.054
0.32	150        (160)	93.75	45.63	45.63	0.058	0.062	0.053
0.56	149        (160)	93.13	43.75	49.38	0.059	0.064	0.055
1.00	147        (160)	91.88	45.63	46.25	0.058	0.061	0.056
1.80	146        (160)	91.25*	43.75	47.50	0.061	0.064	0.058
3.20	130        (160)	81.25*	40.63	40.63	0.056	0.059*	0.052

* significant difference (α= 0.05)

Table 5: Number of laid egg ropes per test concentration

Number of laid egg ropes pertest concentration
[µg a.i./L]	control			0.32			0.56			1.00			1.80			3.20
cage	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
emerged female	29	33	62	35	31	66	42	40	82	34	32	66	32	31	63	14	24	38
egg ropes	36	46	82	35	29	64	43	44	87	40	24	64	31	30	61	10	9	19
fertile egg ropes	31	42	73	29	27	56	40	39	79	30	22	52	22	26	48	6	3	9

Tabele 6: Fecundity as dependent on concentration of the test item

Treatm. [µg a.s./L]	control	0.32	0.56	1.00	1.80	3.20
Cage A	1.241	1.000	1.024	1.176	0.969	0.714
Cage B	1.394	0.936	1.100	0.750	0.968	0.375
Mean:	1.318	0.968	1.062	0.963	0.968	0.545*
Std.Dev.:	0.1078	0.0456	0.0539	0.3016	0.0008	0.2399
n:	2	2	2	2	2	2
CV:	8.2	4.7	5.1	31.3	0.1	44.1

Mean: arithmetic mean; Std.Dev.: standard deviation; n: number of replicates; CV: coefficient of variation

* statistical significance (Student-t test for homogeneous variances with Bonferroni-Holm adjustment (Bonferroni T-Test), α = 0.05, one-sided smaller)

(Fecundity rate = number of laid egg ropes per cage/ number of emerged female midges per cage)

Table 7: Fertility as dependent on concentration of the test item

Treatm. [µg a.i./L]	control	0.32	0.56	1.00	1.80	3.20
Cage A	1.069	0.829	0.952	0.882	0.688	0.429
Cage B	1.273	0.871	0.975	0.688	0.839	0.125
Mean:	1.171	0.850*	0.964	0.785*	0.763*	0.277*
Std.Dev.:	0.1440	0.0300	0.0160	0.1378	0.1069	0.2147
n:	2	2	2	2	2	2
CV:	12.3	3.5	1.7	17.6	14.0	77.6

Mean: arithmetic mean; Std.Dev.: standard deviation; n: number of replicates; CV: coefficient of variation

* statistical significance (Student-t test for homogeneous variances with Bonferroni-Holm adjustment (Bonferroni T-Test), α = 0.05, one-sided smaller)

(Fertility rate = number of fertile egg ropes per cage/ number of emerged female midges per cage)

Table 8: Results based on nominal concentrations in µg a.i./L

Endpoints	NOEC		LOEC		EC15		EC50
Generation:	F1	F2	F1	F2	F1	F2	F1	F2
Emergence rate (pooled sex) (95% confidence limits)	1.80	1.00	3.20	1.80	2.26 (1.78 – 2.71)	> 3.20	> 3.20	> 3.20
development rate (pooled sex) (95% confidence limits)	1.00	≥ 3.20	1.80	> 3.20	2.65 (1.09 – 3.04)	> 3.20	> 3.20	> 3.20
(males) (95% confidence limits)	1.80	1.80	3.20	3.20	2.95 (n.d.)	> 3.20	> 3.20	> 3.20
(females) (95% confidence limits)	1.00	≥ 3.20	1.80	> 3.20	2.42 (1.85 – 2.73)	> 3.20	> 3.20	> 3.20
Fecundity (95% confidence limits)	1.80	-	3.20	-	n.d.1)	-	n.d.1)
Fertility	0.56	-	1.00	-	0.35 (0.012 – 0.69)	-	1.87 (1.10 – 6.72.)

1) No meaningful concentration response was found.

Table 1. Validity criteria for OECD 218

Criterion from the guideline	Outcome	Validity criterion fulfilled
The emergence in the control must be at least 70% at the end of the test.	F1: 86.25% F2: 96.25%	yes
C. riparius emergence to adults from control vessels should occur between 12 and 23 d after their insertion into the vessels.	F1: day 13 - 21 F2: day 14 - 22	yes
At the end of the test, pH and the dissolved oxygen concentration should be measured in each vessel. The oxygen concentration should be at least 60% of the air saturation value (ASV) at the temperature used and the pH of the overlying water should be in the 6-9 range in all test vessels.	Oxygen: 7.9 - 8.8 mg/L pH: 8.4 - 8.9	yes
The water temperature should not differ by more than ± 1.0 °C.	20.1 - 20.6 °C	yes

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

The lowest derived NOEC (28 d) was 0.56 µg a.i./L (nominal) based on on fecundity in a life-cycle toxicity test (OECD 233) with Chironomus riparius.

Description of key information

NOEC (28 d) = 0.56 µg/L (nominal, OECD 233, Chironomus riparius)

Additional information

Three GLP studies are available assessing the toxicity of 3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin on sediment dwelling Chironomus riparius. All studies were performed with spiked water.

The key study assessed the effects of life-long exposure of chemicals to the freshwater dipteran Chironomus riparius, fully covering the first generation and the early part of the second generation following OECD test guideline 233.

First instar larvae of Chironomus riparius were exposed in a static test system for 28 days to initial nominal concentrations in the overlying medium ranging from 0.32 to 3.2 µg a.i./L.

Emerged adult midges of F1 generation were transferred with the test vessels into the breeding cages and released inside the breeding cages. Laid egg ropes from a chosen day around day 19 of the study were used to hatch F2 generation. Subsequently first instar larvae of F2 generation were exposed in a static water-sediment system for 28 days.

As endpoints, the total number of adults emerged (for both 1st and 2nd generation), the development rate (for both 1st and 2nd generation), sex ratio of fully emerged adults (for both 1st and 2nd generation), the number of egg ropes per female (1st generation only) and the fertility of the egg ropes (1st generation only) were recorded. At the beginning of the exposure period (nearly one hour after spiking) analyses reflect high recoveries of thiacloprid for F1-generation and F2-generation in the overlying water of all test concentrations from 77 % to 103 % (mean 89 %) and from 78 % to 94 % (mean 88 %) were found, thus all results and reporting are based on nominal concentrations.

For F1-generation start of emergence was on day 13 for the controls and test concentrations from 0.32 to 1.80 µg a.i./L. The start of emergence was reduced for two days at test concentration of 3.20 µg a.i./L. Statistical significance on emergence rate (pooled sex) was evaluated for 3.20 µg a.i./L, resulting in an NOEC of 1.80 µg a.i./L. For the development rate pooled sex a statistically significance was evaluated at test concentration with emergence of 1.80 and 3.20 µg a.i./L, resulting in an NOEC of 1.00 µg a.i./L.

For F2-generation start of emergence was on day 14 for the controls and test concentrations from 0.32 to 1.80 µg a.i./L. The start of emergence was reduced for one day at test concentration of 3.20 µg a.i./L. 96.3 % of the inserted larvae maturated to adults in the controls after 28 days. Statistical significance on emergence rate (pooled sex) was evaluated for test concentration of 1.80 and 3.20 µg a.i./L, resulting in an NOEC of 1.00 µg a.i./L. For the development rate of pooled sex no statistically significance was evaluated, only for development rate of male midges significance was evaluated at the highest test concentration with emergence of 3.20 µg a.i./L, resulting in an NOEC of 1.80 µg a.i./L.

Statistical significance (a= 0.05) on fecundity rate was evaluated for test concentration of 3.20 µg a.i./L, resulting in an NOEC of 1.80 µg a.i./L. The lowest derived NOEC (28) was found for the endpoint fertility and is 0.56 µg a.i./L.

A second study was performed to to assess potential effects on fecundity and fertility of the chosen test concentrations in comparison to the control in C. riparius, fully covering the first generation and the oviposition phase. The study was performed as a follow up to the key full life cycle study (M-493340-01-1) investigating the same test item with a special focus on fecundity and fertility. First instar larvae were exposed in a static test system for 28 days to nominal concentrations (spiked water application) of 0.32 and 0.56 µg a.i./L of a water-sediment system.

Emerged adult midges of F1 generation were transferred with the test vessels into the breeding cages and released inside the breeding cages. All egg ropes laid on the water surface were collected daily. Any other abnormal observations of adult midges inside the breeding cages or the visual appearance of laid egg ropes were recorded. At the beginning of the exposure period (nearly one hour after spiking) analyses reflect high recoveries of thiacloprid in the overlying water of all test concentrations from 99 % to 102 % (mean 101 %) were found, thus all results and reporting are based on nominal concentrations of thiacloprid in the overlying water, expressed in µg a.i./L. After 28 days 15 % to 16 % (mean 16 %) were detected. No statistical significance (α = 0.05) on emergence rate and development rate (males, females and pooled sex) was evaluated for 0.32 and 0.56 µg a.i./L, resulting in an NOEC of ≥ 0.56 µg a.i./L.

In a third study performed according to a proposal for a BBA-Guideline, first instar larvae were exposed to the nominal initial test concentrations between 0.32 and 10 µg a.i./L for 28 days. The test vessels were observed at least three times per week to make a visual assessment of any behavioral differences between the treated conditions and the control. The sex, time and number of emerged adults were recorded daily during the period of emergence. At test concentrations higher than 1.8 µg a.i./L, no adult midges emerged. The day of first emergence was only postponed at the highest test concentration with successful emergence (1.8 µg a.i./L initial nominal concentration). The derived EC10 was 1.66 µg a.i./L based on emergence and nominal concentrations.