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Sediment toxicity

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Reference
Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016_2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
SEDIMENT
- Concentrations: 100-1000 mg/kg dw
- Sampling interval: 0 - 28 d
- Sample storage before analysis: same day

PORE WATER
- Concentrations: =< 0.4 mg/L
- Sampling interval: 0 - 28 d
- Sample storage before analysis: same day

OVERLYING WATER
- Concentrations: =< 0.4 mg/L
- Sampling interval: 0 - 28 d
- Sample storage before analysis:same day
Vehicle:
yes
Remarks:
Acetone. A solvent control was added.
Details on sediment and application:
Artificial soil substratum as described in the guideline.
Per replicate 12 g sand was spiked with 3 mL acetone stock. After evaporation of the acetone the coated sand was mixed with sediment, which was corrected for the amount of sand used for spiking. Care was taken to ensure that TPS 32 (polysulfides, di-tert-dodecyl) was thoroughly and evenly distributed within the sediment. A bulk amount was prepared and distributed over the replicates. Each replicate contained a total amount of wet sediment of 85 g, consisting of 12 g treated sand and 73 g OECD medium . This corresponds with approximately 60 g dry sediment per vessel . The solvent-control was treated in the same manner as was the TPS 32 (polysulfides, di-tert-dodecyl) test concentration.
Test organisms (species):
Chironomus riparius
Details on test organisms:
In-house laboratory culture with a known history.The entire life cycle lasts approximately 4 weeks at a temperature of ca. 20°C. The fertilized egg masses hatch 2 to 3 days after placing in water.
Thereafter the larvae undergo four stages (L1-L4). L2 larvae emigrate to the sediment. After the fourth stage, the larvae change into pupae. Immediately after the pupa moves to the surface of the water the midge emerges from the pupal skin. The emergence of male midges starts earlier than that of the females.
Females are a little larger and darker than males. Males have plumose antennae.
Mating occurs in swarming. After 1-2 days the female midge lays an egg mass, 2 to 3 days later a second, but smaller, egg mass can be produced. Midges have a life span of four to eight days.

Breeding
Start of each batch Egg masses are used to start a new batch.
Type of culture Continuous culture.
Feeding Pelleted fish food (Cyprico Crumble Excellent, Coppens International BV, Helmond, The Netherlands), at least three times a week.
Conditions Comparable to the test conditions

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)
Hardness:
214 - 268 mgCaCO3/L
Test temperature:
19 - 21 °C
pH:
7.6 - 8.5
Dissolved oxygen:
5.0 - 9.2 mgO2/L
Salinity:
NA
Ammonia:
0 - 3 mg/L
Conductivity:
NA
Nominal and measured concentrations:
Nominal: 0 - 100 - 180 - 320 - 560 - 1000 mg/kg dw
Measured: 0 - 69- 125 - 224 - 385 - 741 mg/kg dw
Details on test conditions:
TEST SYSTEM
A layer of ca. 1.5 cm of spiked sediment was added to each vessel. Thereafter test water (6 cm) was added to the sediment without considerable disturbance of the sediment. The ratio overlying water: sediment was 4:1. Test vessels were covered with a nylon mesh and left under test conditions for an equilibrium period of approximately 48 hours.A layer of ca. 1.5 cm of spiked sediment was added to each vessel. Thereafter test water (6 cm) was added to the sediment without considerable disturbance of the sediment. The ratio overlying water: sediment was 4:1. Test vessels were covered with a nylon mesh and left under test conditions for an equilibrium period of approximately 48 hours.
The test was performed in a climate room with complete control on temperature and light regime (Photoperiod: 16 hour daily). The test vessels were placed at random in fume cupboards within this room.

EXPOSURE REGIME
Four to five days before start of the test (day 0) egg masses were taken from the culture and deposited into small vessels in culture medium. Twenty larvae of the first larval stage (2 to 3 days old) were allocated randomly to each test vessel with a pipette. During the addition of the larvae and for approximately 24 hours thereafter, aeration was stopped.

RENEWAL OF OVERLYING WATER
Three times a week. If applicable, evaporated water was replaced by Milli-RO water.

OVERLYING WATER CHARACTERISTCS
Adjusted ISO medium
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L




CHARACTERIZATION OF ARTIFICIAL;SEDIMENT
Sphagnum peat 5 %
Kaolin clay 20 %
Industrial (silver) sand 75 %



VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Nominal: 0 - 100 - 180 - 320 - 560 - 1000 mg/kg dw
- Range finding study: yes 1 - 10 - 100 -1000 mg/kg
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
741 mg/kg sediment dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
emergence rate
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
> 741 mg/kg sediment dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
emergence rate
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
385 mg/kg sediment dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
development rate
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
> 741 mg/kg sediment dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
development rate
Details on results:
- No. of emerged male and female midges (per vessel and per day): detailed tables provided in study report. Since there was no statistically significant effect on sex ratio, pooled data on male and female chironomids were used for further statistical calculations.
- No. of pupae failing to emerge (per vessel and per day): detailed tables provided in study report.
- Percent emergence per replicate and test concentration see table below.
- Mean development rate of fully emerged midges (per replicate and treatment rate; male and female midges pooled): see table below.
- Mean individual dry weight of larvae (per larvae and per instar):

- Total biomass per replicate:
- Morphological abnormalities:
- Behavioural abnormalities:
- Other biological observations:

Emergence rate of chironomids during the final test

Concentration

TPS 32 (polysulfides, di-tert-dodecyl)

(mg/kg d.w.)

Total

Introduced

Emerged

Not-emerged

% Not-emerged

% Emerged

Blank-control

80

79

1

1.2

98.8

Solvent -control

80

73

7

8.7

91.3

69

82

79

3

3.7

96.3

125

80

75

5

6.3

93.7

224

80

76

4

5.0

95.0

385

80

78

2

2.5

97.5

741

80

75

5

6.3

93.7

 

Development rate of chironomids after 28 days in the final test

Concentration

TPS 32 (polysulfides, di-tert-dodecyl)

(mg/kg d.w.)

Mean

Std. Dev.

n

% Inhibition

compared to the solvent-control

Blank-control

0.061

0.0017

4

 

Solvent -control

0.060

0.0016

4

 

69

0.062

0.0013

4

-3.5

125

0.061

0.0015

4

-1.8

224

0.060

0.0027

4

0.2

385

0.060

0.0015

4

0.8

741

0.058

0.0003

4

4.0

Validity criteria fulfilled:
yes
Conclusions:
No statistically significant difference in emergence was observed at and below 741 mg/kg d.w. (NOEC) compared to the solvent-control.
No statistically significant effect on development rate was observed at and below 385 mg/kg d.w. (NOEC) compared to the solvent-control.
Executive summary:

The study procedure was based on OECD Guideline No. 218 (2004).

 

The batch of TPS 32 (polysulfides, di-tert-dodecyl) tested was a clear yellow liquid. Based on the results of the range-finding test, a final test was performed with concentrations ranging from 100 to 1000 mg/kg d.w., increasing with a factor of 1.8. Furthermore, a blank-control and a solvent-control were included.

 

The test concentrations were prepared by mixing spiked sand, after evaporation of the solvent, with sediment. A bulk amount was prepared and distributed over the replicates. In total 80 larvae per group (20 per replicate/4 replicates) were exposed to the controls (blank and solvent-control) and theTPS 32 (polysulfides, di-tert-dodecyl) concentrations. The total test period was 28 days. On days 0 and 28 samples for analysis were taken from overlying water, pore water and sediment from the concentrations.

 

The results of analysis at the start and at the end of the final test showed that the measured concentrations in sediment were not in agreement with the nominal concentrations. The mean measured concentrations were 69, 125, 224, 385 and 741 mg/kg d.w. at nominal concentrations of 100, 180, 320, 560 and 1000 mg/kg d.w., respectively. Final endpoints were estimated in terms of mean measured concentrations.

 

The amount of TPS 32 (polysulfides, di-tert-dodecyl) recovered in the sediment layer on day 0 was between 73% and 80% of the applied amount of TPS 32 (polysulfides, di-tert-dodecyl) and between 64% and 69% at day 28.

The amount of TPS 32 (polysulfides, di-tert-dodecyl) recovered in the overlying water on day 0 and day 28 was below 0.2% the applied amount of TPS 32 (polysulfides, di-tert-dodecyl).

The amount of TPS 32 (polysulfides, di-tert-dodecyl) recovered in the pore water on day 0 and day 28 was below 0.1% the applied amount of TPS 32 (polysulfides, di-tert-dodecyl).

The mass balance was between 73% and 80% at the start of the final test and between 64% and 69% at the end of the final test (based on mean values of replicates).

 

Since, there was no statistically significant effect on sex ratio, pooled data on male and female chironomids were used for further statistical calculations.

 

Emergence in the blank-control and solvent-control was 99% and 91%, respectively. The difference on emergence rate of chironomids between the blank-control and the solvent-control was statistically significant. On development of chironomids no statistically significant effect between the blank-control (0.061day-1) and the solvent-control (0.060day-1) was observed. Therefore, the solvent-control was used for further statistical evaluations.

 

In both control groups and in the concentrations up to and including 385 mg/kg d.w. the first midges emerged on day 15. At 741 mg/kg d.w. the first midges emerged on day 16.

No statistically significant differences in emergence were observed in any of the concentrations tested compared to the solvent-control (Chi²-2 x 2 Test with Bonferroni Correction, a = 0.050; one-sided greater).Thus, the NOEC was set at the highest concentration tested, i.e. 741 mg/kg d.w.

 

No statistically significant differences in development rate were observed in concentrations up to and including 385 mg/kg d.w. compared to the solvent-control (NOEC). Whereas the effect in the highest concentration tested, i.e. 741 mg/kg d.w.was statistically significant compared to the solvent-control (Williams Multiple Sequential t-test procedure, a = 0.050, one-sided smaller).

 

No pupae that failed to emerge were observed at the end of the test. No egg masses were observed during the test in any of the vessels and no behavioural differences in test substance exposed larvae compared to the controls were recorded.

Description of key information

The study procedure was based on OECD Guideline No. 218 (2004) aiming at evaluating effects of chemical substances on emergence and development of midges (chironomids) from sediment contaminated with a range of test substances concentrations (5).

The study was carried out according to GLP with no deviations from the OECD TG 218. Results in the table below are calculated according to mean measured test concentrations. Among the 2 measured endpoints, emergence and development, the latter is more sensible and will be used a key value for PNEC derivation and risk assessment.

Parameter

TPS 32 (polysulfides, di-tert-dodecyl) (mg/kg d.w.)

 

NOEC

EC10

EC50

 

Emergence rate

Value

741

> 741

> 741

 

Development rate

Value

385

> 741

> 741

 

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:
385 mg/kg sediment dw

Additional information

The recently (2017) obtained data on chronic toxicity of polysulfides, di-tert-dodecyl to sediment organisms (emergence and development of midges larvae) provided an NOEC and hence allows derivation of a PNECsed.