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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Sampling for Measurement of Test Concentrations
Duplicate samples for analysis were taken from all test concentrations and the control.
Sampling Frequency: Weekly, i.e. at the beginning and at the end of three intervals of 48 hours (days 0-2, 14-16, 21-23) and two intervals of 72 hours (nominal days 9-12, 30-33)
Volume: 2.0 mL from the total solution freshly prepared and from the old solutions in each vessel
Storage: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
Vehicle:
no
Test organisms (species):
Pimephales promelas
Details on test organisms:
Test System
- Species: Fathead minnow (Pimephales promelas, Teleostei Cyprinidae) Rafinesque.
- Source: In house culture.

Holding of the Brood Stock
- Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
- Measurements: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous.
- Water quality parameters: Will be kept within the optimum limits for the respective fish species.
- Ratio male/female: 1:2
- Spawning tank: The spawning tank is equipped with a substrate (pvc-tube), which enables collection of the fertilised eggs.
- Feeding brood stock: Frozen brine shrimp Nauplii and pelleted fish food (SDS 400, Coppens International bv, Helmond, The Netherlands).
Time of fertilisation: Spawning starts the following day approximately 1 to 2 hours after lights have been switched on.

Testing Strategy and Experimental Design
Analytical Feasibility Test
The project started with an analytical feasibility test to check whether the test concentrations for the main test could be analytically monitored and to check stability over the renewal intervals. Saturated solutions (SS) were individually prepared at a loading rate of 10 mg/L. Two solutions at a volume of 1 litre and two other at a volume of 10 litres. In addition, a 10-fold dilution was prepared from each of the solutions (10% SS). All end solutions were prepared and incubated under similar test conditions as indicated for the main study but without organisms and applying continuous aeration. Triplicate samples for analyses were taken from each of the solutions at the start and the end of a 48-hour incubation period.

Early Life Stage Test
- Test type: Semi-static
- Frequency of renewal: Three times a week, starting on day 2 of the study.
- Test duration: 33 days
- Introduction egg: Before cleavage of the blastodisc commenced (approximately 2-4 hours after fertilisation).
- Test vessels Embryonic phase: Petri-dishes (50 mL).
Larval phase: Stainless steel vessels (1.7 L).
- Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.

The experiment (nominal day 0) started with fresh and healthy fertilised fathead minnow eggs per test group. The fertilised eggs were randomly distributed and divided equally over three petri-dishes. Each petri-dish contained 20 eggs in 50 mL test medium. On day 7 the hatched larvae were transferred to stainless steel test vessels.
- Light period: 16 h photoperiod daily, between 866 and 982 lux.
- Feeding: Embryonic phase: no feeding.
Newly hatched larvae: Brachionus suspensions.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d
Test temperature:
23.7 - 26.0°C (min-max)
pH:
7.5 - 8.1 (min-max)
Dissolved oxygen:
5.1 - 9.4 mg/L (min-max)
Nominal and measured concentrations:
Nominal: control, 4.6, 10, 22, 46 and 100% of a saturated solution prepared at a loading rate of 10 mg/L.
Measured: as the substance is poorly soluble, only the highest tested concentration was analytically measured, at 5 occasions (fresh/old). The arithmetical mean of the geometrical means was 1.1 µg/L.
Details on test conditions:
TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): petri-dishes (50 mL)
- Test vessel: stainless steel vessels (1.7 L)
- Type (delete if not applicable): open
- Renewal rate of test solution: three times a week (semi-static) with freshly prepared stock solution
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Adjustment of pH: not needed
- Photoperiod: 16L:8D
- Light intensity: 866 -982 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Hatching rate
- Larval survival rate at the end of the test
- Larval weight at the end of the test
- Larval length at the end of the test


POST-HATCH DETAILS
- Begin of post-hatch period:
- No. of hatched eggs (alevins)/treatment released to the test chamber:
- Release of alevins from incubation cups to test chamber on day no.:

FERTILIZATION SUCCESS STUDY
- Number of eggs used:
- Removal of eggs to check the embryonic development on day no.:
Reference substance (positive control):
no
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
> 1.1 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
number hatched
Remarks on result:
other: No effect up to solubility limit
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
> 1.1 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: No effect up to solubility limit
Key result
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
> 1.1 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
length
Remarks on result:
other: No effect up to solubility limit
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
> 1.1 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
weight
Remarks on result:
other: No effect up to solubility limit
Details on results:
Measured Concentrations
Table 12 shows the mean concentrations measured in the samples taken from the undiluted SS prepared at a loading rate of 10 mg/L in fresh and old solutions during the test period. Mean measured concentrations in samples taken from freshly prepared SS solutions ranged between 0.67 and 3.3 µg/L. These concentrations, with some variations, were considered to present the limit of solubility in test medium. During the renewal periods measured concentrations decreased by 40% or more. A scientifically sound explanation for the decrease cannot be given, but the most plausible explanation would be related to adsorption to or by the test organisms, adsorption to food and/or adsorption to other parts of the test system. In addition, it might even be that actual solubility in test medium under the conditions of this study was even below the initial concentration in freshly prepared solutions. It was concluded that the average exposure concentration of 1.1 µg/L, as measured in the saturated solution, represented the maximum concentration achievable under the test conditions with this poorly soluble test item.

Embryonic Survival
The overall survival of embryos at the end of hatching was 96% for the control. Hence, the validity criterion of >70% survival until hatching was complete was met. Hatching percentage in the test item treated groups was comparable to the control and ranged from 94 to 100% without a relation to the applied treatment. No statistically significant differences when compared to the control were observed at any of the concentrations tested. Hence, the NOEC was at or above the saturation level of the test item in medium (=100% SS) corresponding to an average exposure concentration of 1.1 µg/L (Table 1).

Larval Survival and Development
The mean post-hatch larval survival was 97% for the control. Hence, the validity criterion for post-hatch survival of at least 75% was met. Post-hatch survival in the test item treatment groups was comparable to the control and ranged from 95 to 100% without a relation to the applied treatment (Table 2). There were no statistically significant differences for post-hatch survival between the control and the different Bis(piperidionpiperidinothiocarbonyl) hexasulfide treatments. Hence, the NOEC for survival was at or above the saturation level of the test item in medium (=100% SS) corresponding to an average exposure concentration of 1.1 µg/L. No significant effects on e.g. swimming behaviour or appearance were observed in the control treatment or any of the test item treatment groups during the course of the study. The few symptoms that were observed were not test item related and considered to derive from natural variation as they were also observed in the control treatment.

Effects on Larval Growth
Body lengths and body weights measured at the end of the test period are summarised in Tables 3 and 4. There were no statistically significant differences for both body weight and length between the control and the different Bis(piperidionpiperidinothiocarbonyl) hexasulfide treatments. Hence, the NOEC for growth (mean body length and weight of larvae) was at or above the saturation level of the test item in medium (=100% SS) corresponding to an average exposure concentration of 1.1 µg/L.

More detailed data are available from Table 5 to Table 11.
Reported statistics and error estimates:
The following statistical procedures were used to determine the NOEC values for embryonic and larval survival, body length and weight. Hatchability (embryonic survival) and larval survival:

• Differences between treatments and the control: Chi²-2 x 2 table test with Bonferroni Correction (a=0.05, one-sided greater) after qualitative trend analysis by contrasts (monotonicity of concentration/response).
NOEC for body length and body weight:
• Data distribution: Shapiro-Wilk´s Test
• Homogeneity of variance: Levene´s Test (with Residuals)
• Dunnett’s Multiple t-test Procedure (a=0.05, one-sided smaller) after trend analysis by contrasts (monotonicity of concentration/response).

All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 1: Mean hatching rate per treatment on day 8

Bis(piperidinothiocarbonyl) hexasulfide

%SS prep. at 10 mg/L

Total eggs

introduced

Hatched

Not hatched

% Post-hatch

mortality

 Control

80

77

3

3.8

4.6

80

80

0

0.0

10

80

77

3

3.8

22

80

78

2

2.5

46

80

77

3

3.8

100

80

75

5

6.3

 

Table 2: Mean post-hatch mortality at the end of exposure (day 33)

Bis(piperidinothiocarbonyl) hexasulfide

%SS prep. at 10 mg/L

Total1

introduced

Survived

Dead

% Post-hatch

mortality

 Control

77

75

2

2.6

4.6

80

78

2

2.5

10

77

74

3

3.9

22

78

74

4

5.1

46

77

74

3

3.9

100

75

75

0

0.0

1. Total number of hatched larvae

Table 3: Mean body weight (mg) and percentage of weight reduction at the end of the exposure (day 33)

 

Bis(piperidinothiocarbonyl) hexasulfide

%SS prep. at 10 mg/L

Mean
(mg)

Std. Dev.

n

%Reduction

 Control

76.50

12.71

4

4.6

79.75

8.42

4

-4.2

10

77.50

4.80

4

-1.3

22

73.75

4.57

4

3.6

46

77.50

2.65

4

-1.3

100

72.00

2.83

4

5.9

 

Table 4: mean length (mm) and percentage length reduction at the end of the exposure (day 33)

 

Bis(piperidinothiocarbonyl) hexasulfide

%SS prep. at 10 mg/L

Mean
(mm)

Std. Dev.

n

%Reduction

 Control

21.78

0.75

4

 

4.6

21.38

0.54

4

1.8

10

21.02

0.41

4

3.4

22

20.92

0.40

4

3.9

46

21.35

0.31

4

2.0

100

21.13

0.74

4

3.0

 

Table 5: Effect parameters

 

Parameter

LOEC

NOEC

EC50

Embryonicsurvival

Value1

> 1.1 µg/L

=1.1µg/L

> 1.1 µg/L

>100% SS

=100% SS

>100% SS

 

 

 

 

 

Post-hatch

Value2

> 1.1 µg/L

=1.1µg/L

> 1.1 µg/L

survival

 

>100% SS

=100% SS

>100% SS

 

 

 

 

 

Growth

Value2

> 1.1 µg/L

=1.1µg/L

> 1.1 µg/L

(weight)

 

>100% SS

=100% SS

>100% SS

 

 

 

 

 

Growth

Value2

> 1.1 µg/L

=1.1µg/L

> 1.1 µg/L

(length)

 

>100% SS

=100% SS

>100% SS

 

 

 

 

 

1- for day 8 of exposure,2- for day 33 of exposure

Table 6: Analytical results of the samples taken during the ELS test

%SS prepared at 10 mg/L

Mean measured concentrations (µg/L)

Mean

Day

0-33

(µg/L)

Day 0

fresh

Day 2

old

Day 9

fresh

Day 12

old

Day 14

fresh

Day 16

old

Day 21

fresh

Day 23

old

Day 30

fresh

Day 33

old

100

1.3

<0.69

1.8

0.15

2.1

1.3

3.3

1.6

0.67

<0.19

1.1

Note that all ‘<’ values correspond to < LOD on the respective day of analysis. Half of the LOD value was used to calculate the average exposure concentration over 0-33 d. Result of fresh samples represent mean of duplicate samples. Result of old samples are means of singular samples taken from 4 pooled replicates.

Table 7: Survival of Fish Embryos of the Control and the Bis(piperidinothiocarbonyl) hexasulfide Treated Groups

Bis(piperidinothiocarbonyl) hexasulfide

%SS prep at
10 mg/L

Replicate

Number of eggs

at start

Nominal day

1

2

3

4

5

D

A

D

A

D

A

D

A

D

A

Control

A

20

2

18

1

17

0

17

0

17

0

7

 

B

20

0

20

0

20

0

20

0

20

0

5

 

C

20

0

20

0

20

0

20

0

20

0

7

 

D

20

0

20

0

20

0

20

0

20

0

14

4.6

A

20

0

20

0

20

0

20

0

20

0

17

 

B

20

0

20

0

20

0

20

0

20

0

12

 

C

20

0

20

0

20

0

20

0

20

0

2

 

D

20

0

20

0

20

0

20

0

20

0

11

10

A

20

1

19

0

19

0

19

0

19

0

12

 

B

20

0

20

0

20

0

20

0

20

0

4

 

C

20

0

20

0

20

0

20

0

20

0

12

 

D

20

2

18

0

18

0

18

0

18

0

6

22

A

20

0

20

0

20

0

20

0

20

0

6

 

B

20

0

20

0

20

0

20

0

20

0

2

 

C

20

1

19

0

19

0

19

0

19

0

14

 

D

20

1

19

0

19

0

19

0

19

0

14

46

A

20

1

19

0

19

0

19

0

19

0

13

 

B

20

2

18

0

18

0

18

0

18

0

3

 

C

20

0

20

0

20

0

20

0

20

0

7

 

D

20

0

20

0

20

0

20

0

20

0

10

100

A

20

1

19

0

19

0

19

0

19

0

10

 

B

20

2

18

0

18

0

18

0

18

0

14

 

C

20

1

19

0

19

0

19

0

19

0

14

 

D

20

1

19

0

19

0

19

0

19

0

15

D = dead, A= alive

 

Survival of Fish Embryos of the Control and the Bis(piperidinothiocarbonyl) hexasulfide Treated Groups (continued)

Bis(piperidinothiocarbonyl) hexasulfide

%SS prep at
10 mg/L

Replicate

Number of eggs

at start

Nominal day

% Embryonic survival

6

7

8

D

A

D

A

D

A

Control

A

20

 

-1

 

-1

 

-1

85

 

B

20

 

-1

 

-1

 

-1

100

 

C

20

 

-1

 

-1

 

-1

100

 

D

20

 

-1

 

-1

 

-1

100

4.6

A

20

 

6

 

-1

 

-1

100

 

B

20

 

4

 

-1

 

-1

100

 

C

20

 

-1

 

-1

 

-1

100

 

D

20

 

-1

 

-1

 

-1

100

10

A

20

 

-1

 

-1

 

-1

100

 

B

20

 

1

 

-1

 

-1

100

 

C

20

 

4

 

1

 

-1

100

 

D

20

 

2

 

-1

 

-1

100

22

A

20

 

1

 

-1

 

-1

100

 

B

20

 

-1

 

-1

 

-1

100

 

C

20

 

1

 

-1

 

-1

100

 

D

20

 

1

 

-1

 

-1

100

46

A

20

 

5

 

-1

 

-1

100

 

B

20

 

1

 

-1

 

-1

100

 

C

20

 

1

 

-1

 

-1

100

 

D

20

 

-1

 

-1

 

-1

100

100

A

20

 

-1

 

-1

 

-1

100

 

B

20

 

6

 

1

 

-1

100

 

C

20

 

1

 

-1

 

-1

100

 

D

20

 

3

 

-1

 

-1

100

D = dead, A= alive,1– all embryos hatched in this replicate

Table 8: Hatching and post-hatch survival of larvae of the control and the test item treated groups

Bis(piperidinothiocarbonyl)
hexasulfide

%SS prep at
10 mg/L

Replicate

Number of

eggs at day 0

Nominal day

4

5

6

7

A

D

A

D

A

D

A

D

Control

A

20

-

-

10

0

17

0

17

0

B

20

-

-

15

0

20

0

20

0

C

20

-

-

13

0

20

0

20

0

D

20

-

-

6

0

20

0

20

0

4.6

A

20

-

-

3

0

14

0

20

0

B

20

-

-

8

0

16

0

20

0

C

20

-

-

18

0

20

0

20

0

D

20

-

-

9

0

19

1

19

0

10

A

20

-

-

7

0

19

0

19

0

B

20

-

-

16

0

19

0

20

0

C

20

-

-

8

0

16

0

201

0

D

20

-

-

12

0

16

0

18

0

22

A

20

-

-

14

0

19

0

20

0

B

20

-

-

18

0

20

0

20

0

C

20

-

-

5

0

18

0

19

0

D

20

-

-

5

0

18

0

19

0

46

A

20

-

-

6

0

14

0

19

0

B

20

-

-

15

0

17

0

18

0

C

20

-

-

13

0

19

0

20

0

D

20

-

-

10

0

20

0

20

0

100

A

20

-

-

9

0

19

0

19

0

B

20

-

-

4

0

12

0

181

0

C

20

-

-

5

0

18

0

19

0

D

20

-

-

4

0

16

0

19

0

-= no hatched larvae, A= Alive, D=Dead, 1.One embryo alive but not hatched yet

Table 9: Post-hatch survival of larvae of the control and the test item treated groups (Day 8-14)

 



Bis(piperidinothiocarbonyl)
hexasulfide

%SS prep at
10 mg/L

Replicate

Mortalities at nominal day

Alive1

8

9

10

11

12

13

14

14

Control

A

0

0

0

0

0

0

0

17

 

B

0

0

0

0

0

0

0

20

 

C

0

0

0

0

0

0

0

20

 

D

0

0

1

0

0

0

0

19

4.6

A

0

0

0

0

0

0

0

20

 

B

0

0

0

0

0

0

0

20

 

C

0

0

0

0

0

0

0

20

 

D

0

1

0

0

0

0

0

18

10

A

0

0

0

0

0

0

0

19

 

B

0

0

0

0

0

0

0

20

 

C

0

0

0

0

0

0

0

20

 

D

0

0

0

0

0

0

0

18

22

A

1

0

0

0

0

0

1

18

 

B

0

0

0

0

0

0

0

20

 

C

0

0

0

0

0

0

0

19

 

D

0

0

0

1

0

0

0

18

46

A

0

1

0

0

0

0

0

18

 

B

0

0

0

0

0

0

0

18

 

C

1

0

0

0

0

0

0

19

 

D

0

0

0

0

0

0

0

20

100

A

0

0

0

0

0

0

0

19

 

B

0

0

0

0

0

0

0

18

 

C

0

0

0

0

0

0

0

19

 

D

0

0

0

0

0

0

0

19

1. Counted number of living fish

 

Table 10: Post-hatch survival of larvae of the control and the test item treated groups (Day 15-21)

 



Bis(piperidinothiocarbonyl)
hexasulfide

%SS prep at
10 mg/L

Replicate

Mortalities at nominal day

Alive1

15

16

17

18

19

20

21

21

Control

A

0

0

0

0

0

0

0

 16*

 

B

0

0

0

0

0

0

0

20

 

C

0

0

0

0

0

0

0

20

 

D

0

0

0

0

0

0

0

19

4.6

A

0

0

0

0

0

0

0

20

 

B

0

0

0

0

0

0

0

20

 

C

0

0

0

0

0

0

0

20

 

D

0

0

0

0

0

0

0

18

10

A

0

0

0

0

0

0

0

19

 

B

0

0

0

0

0

0

1

19

 

C

0

1

0

0

0

1

0

18

 

D

0

0

0

0

0

0

0

18

22

A

0

0

0

0

0

0

0

18

 

B

0

0

0

0

0

0

0

20

 

C

0

0

0

0

0

0

0

19

 

D

0

0

0

0

0

0

0

18

46

A

0

0

0

0

0

0

0

18

 

B

0

0

0

0

0

0

0

18

 

C

0

0

0

0

0

0

0

19

 

D

1

0

0

0

0

0

0

19

100

A

0

0

0

0

0

0

0

19

 

B

0

0

0

0

0

0

0

18

 

C

0

0

0

0

0

0

0

19

 

D

0

0

0

0

0

0

0

19

1. Counted number of living fish, * One fish was missing

Table 11: Post-hatch survival of larvae of the control and the test item treated groups (Day 22-33)

 



Bis(piperidinothio-
carbonyl)
hexasulfide

%SS prep at
10 mg/L

Replicate

Mortalities at nominal day

Alive1

22

23

24

25

26

27

28

29

30

21

32

33

33

Control

A

0

0

0

0

0

0

0

0

0

0

0

0

16

 

B

0

0

0

0

0

0

0

0

0

0

0

0

20

 

C

0

0

0

0

0

0

0

0

0

0

0

0

20

 

D

0

0

0

0

0

0

0

0

0

0

0

0

19

4.6

A

0

0

0

0

0

0

0

0

0

0

0

0

20

 

B

0

0

0

0

0

0

0

0

0

0

0

0

20

 

C

0

0

0

0

0

0

0

0

0

0

0

0

20

 

D

0

0

0

0

0

0

0

0

0

0

0

0

18

10

A

0

0

0

0

0

0

0

0

0

0

0

0

19

 

B

0

0

0

0

0

0

0

0

0

0

0

0

19

 

C

0

0

0

0

0

0

0

0

0

0

0

0

18

 

D

0

0

0

0

0

0

0

0

0

0

0

0

18

22

A

0

0

0

0

0

0

0

0

0

0

0

0

18

 

B

0

0

0

0

0

0

0

0

1

0

0

0

19

 

C

0

0

0

0

0

0

0

0

0

0

0

0

19

 

D

0

0

0

0

0

0

0

0

0

0

0

0

18

46

A

0

0

0

0

0

0

0

0

0

0

0

0

18

 

B

0

0

0

0

0

0

0

0

0

0

0

0

18

 

C

0

0

0

0

0

0

0

0

0

0

0

0

19

 

D

0

0

0

0

0

0

0

0

0

0

0

0

19

100

A

0

0

0

0

0

0

0

0

0

0

0

0

19

 

B

0

0

0

0

0

0

0

0

0

0

0

0

18

 

C

0

0

0

0

0

0

0

0

0

0

0

0

19

 

D

0

0

0

0

0

0

0

0

0

0

0

0

19

1. Counted number of living fish

Table 12: Analytical Results of the Samples Taken During the ELS Test

%SS prepared at 10 mg/L

Mean measured concentrations (µg/L)

Mean

Day

0-33

(µg/L)

Day 0

fresh

Day 2

old

Day 9

fresh

Day 12

old

Day 14

fresh

Day 16

old

Day 21

fresh

Day 23

old

Day 30

fresh

Day 33

old

100

1.3

<0.69[WN1] 

1.8

0.15

2.1

1.3

3.3

1.6

0.67

<0.19

1.1

Note that all ‘<’ values correspond to < LOD on the respective day of analysis. Half of the LOD value was used to calculate the average exposure concentration over 0-33 d (see also section 6.1. ‘Data handling’).

Result of fresh samples represent mean of duplicate samples.

Result of old samples are means of singular samples taken from 4 pooled replicates.

Validity criteria fulfilled:
yes
Conclusions:
No adverse effect was observed on fish early-life stages of Pimephales promelas up to the solubility limit of Bis(piperidinothiocarbonyl) hexasulfide.
Executive summary:

The objective of the study was to define the lethal and sub lethal effects of Bis(piperidinothiocarbonyl) hexasulfide on the early life stages of fish in a semi-static system. For this objective the early-life stages of fathead minnow (Pimephales promelas) were exposed to a range of concentrations of the test item dissolved in water for 33 days. Lethal and sub-lethal effects were assessed and compared with control values to determine the various effect concentrations. The study procedures described in this report were based on the OECD guideline for Testing of Chemicals: Guideline No. 210, 2013.

A Saturated Solution (SS) was prepared at a loading rate of 10 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium. Test solutions were renewed three times per week.

The project started with an analytical feasibility test to check whether the test concentrations for the main test could be analytically monitored and to check stability over the renewal intervals. Approximately 2-3 µg/L was measured in samples from the 100% SS solutions. It was concluded that it was possible to measure actual exposure concentrations in a main ELS test to be performed with a range between 4.6 and 100% SS.

A full Early Life Stage (ELS) test was performed with a control group and Bis(piperidinothiocarbonyl) hexasulfide test groups corresponding with 4.6, 10, 22, 46 and 100% of a SS prepared at a loading rate of 10 mg/L (four replicates each, 20 eggs per replicate).The test started by placing fertilised eggs in petri-dishes under semi-static conditions until most eggs had hatched (day 7). After 7 days of exposure, larvae were transferred to stainless steel test vessels and the exposure was continued for a total period of 33 days. During the embryonic and larval phases the fish were observed for effects on development, appearance and swimming behaviour. At the end of the test the fish were measured and weighed. Samples for chemical analysis of the actual Bis(piperidinothiocarbonyl) hexasulfide concentrations were taken from various intervals during the test of both fresh and old solutions.

The results of the study showed no statistically significant effects up to and including the highest test group on any of the end points being embryonic and larval survival and effects on growth (length and weight). Consequently, results of sample analysis reporting were limited to the highest test group.

Mean measured concentrations in samples taken from freshly prepared SS solutions ranged between 0.67 and 3.3 µg/L. These concentrations, with some variations, were considered to present the limit of solubility in test medium. During the renewal periods measured concentrations decreased by 40% or more.

It was concluded that the average exposure concentration of 1.1 µg/L, as measured in the saturated solution, represented the maximum concentration achievable under the test conditions with this poorly soluble test item.

The study met the acceptability criteria prescribed by the study plan and the guidelines and was considered valid.

In conclusion,the present study assessed the possible lethal and sub-lethal effects of Bis(piperidinothiocarbonyl) hexasulfide during the embryonic and early larval development of the fathead minnow. The results led to the following conclusions for Bis(piperidinothiocarbonyl) hexasulfide:

1.        Bis(piperidinothiocarbonyl) hexasulfide did not affect the hatching success (embryonic survival) up to and including 100% SS prepared at a loading rate of 10 mg/L, corresponding to an average exposure concentration of 1.1 µg/L (NOEC).

2.       Bis(piperidinothiocarbonyl) hexasulfide did not affect survival of larvae up to and including 100% SS prepared at a loading rate of 10 mg/L, corresponding to an average exposure concentration of 1.1 µg/L (NOEC).

3.       Bis(piperidinothiocarbonyl) hexasulfide did not cause any significant developmental malformations up to and including 100% SS prepared at a loading rate of 10 mg/L, corresponding to an average exposure concentration of 1.1 µg/L.

4.       Bis(piperidinothiocarbonyl) hexasulfide did not significantly affect the growth (body weight and length) of the exposed larvae up to and including 100% SS prepared at a loading rate of 10 mg/L, corresponding to an average exposure concentration of 1.1 µg/L.

Hence, the overall NOEC of Bis(piperidinothiocarbonyl) hexasulfide for the early life stages of fish equalled 100% SS prepared at a loading rate of 10 mg/L, corresponding to an average exposure concentration of 1.1 µg/L. In conclusion, no adverse effect was observed at the maximum concentration achievable under the test conditions.

Description of key information

No adverse effect was observed atthe maximum concentration achievable under the test conditions.

Key value for chemical safety assessment

Additional information

The objective of the study was to define the lethal and sub lethal effects of dipentamethylene thiuram hexasulfide on the early life stages of fish in a semi-static system. For this objective the early-life stages of fathead minnow (Pimephales promelas) were exposed to a range of concentrations of the test item dissolved in water for 33 days. Lethal and sub-lethal effects were assessed and compared with control values to determine the various effect concentrations. The study procedures described in this report were based on the OECD guideline for Testing of Chemicals: Guideline No. 210, 2013.

A full Early Life Stage (ELS) test was performed with a control group and DPTH test groups corresponding with 4.6, 10, 22, 46 and 100% of a SS prepared at a loading rate of 10 mg/L (four replicates each, 20 eggs per replicate).

Mean measured concentrations in samples taken from freshly prepared SS solutions ranged between 0.67 and 3.3 µg/L. These concentrations, with some variations, were considered to present the limit of solubility in test medium. During the renewal periods measured concentrations decreased by 40% or more. It was concluded that the average exposure concentration of 1.1 µg/L, as measured in the saturated solution, represented the maximum concentration achievable under the test conditions with this poorly soluble test item.

The results led to the following conclusions for DPTH:

1.        DPTHdid not affect the hatching success (embryonic survival) up to and including 100% SS prepared at a loading rate of 10 mg/L, corresponding to an average exposure concentration of 1.1 µg/L (NOEC).

2.      DPTHdid not affect survival of larvae up to and including 100% SS prepared at a loading rate of 10 mg/L, corresponding to an average exposure concentration of 1.1 µg/L (NOEC).

3.       DPTHdid not cause any significant developmental malformations up to and including 100% SS prepared at a loading rate of 10 mg/L, corresponding to an average exposure concentration of 1.1 µg/L.

4.       DPTHdid not significantly affect the growth (body weight and length) of the exposed larvae up to and including 100% SS prepared at a loading rate of 10 mg/L, corresponding to an average exposure concentration of 1.1 µg/L.

Hence, the overall NOEC of DPTH for the early life stages of fish equalled 100% SS prepared at a loading rate of 10 mg/L, corresponding to an average exposure concentration of 1.1 µg/L. In conclusion, no adverse effect was observed atthe maximum concentration achievable under the test conditions.