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Reference
Endpoint:
sediment toxicity: long-term
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:
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: All
- Sampling interval: Day 0, 7 and 28
- Sample storage before analysis: None

PORE WATER
- Concentrations: All
- Sampling interval: Day, 0, 7, 28
- Sample storage before analysis: None

OVERLYING WATER
- Concentrations: All
- Sampling interval: Day 0, 7, 28
- Sample storage before analysis: None




Vehicle:
yes
Details on sediment and application:
Test Sediment
Formulated sediment, as described in Kemble et al. (1999) was used as the test sediment. This formulated sediment is similar to that described in OECD
Guideline 218, but uses alpha-cellulose as its source of organic matter instead of peat moss. Alpha-cellulose was selected by Kemble et al. (1999) as a more standardized source of organic matter than peat moss. The sediment was composed of 0.01% humic acid, 0.99% dolomite, 5% alpha-cellulose,
14% silt and clay (kaolin clay) and 80% industrial quartz sand. The dry constituents of the sediment were mixed in a PK Twinshell® mixer for 20 minutes, and the batch was stored under ambient conditions until used. The pH of the dry sediment was determined prior to use in the test and was within the desired range of 7.0 (± 0.5). The final pH of a 1:1 ratio of the sediment and water was 6.7. A sample of the formulated sediment used in the test was sent to Agvise Laboratories, Northwood, North Dakota, for characterization and analysis of total organic carbon (TOC), and a summary of the sediment characterization is presented in Appendix 6. The percent organic carbon of the sediment was determined to be 2.8%.

Preparation of Test Concentrations
Individual stock solutions were prepared for use in spiking the sediment to prepare each of the five concentrations tested. Test concentrations were not adjusted for the active ingredient of the test substance during preparation, and are based on the test substance as received. A 25-mL primary
stock solution was prepared by mixing a calculated amount of test substance into acetone at a nominal concentration of 200 mg/mL. The primary
stock solution was sonicated for approximately one minute and was stirred on a magnetic stir plate for approximately one hour, and appeared clear
and light yellow with no visible precipitates. Five secondary stock solutions (20 mL each) were prepared in acetone at nominal concentrations of 6.3,
12.5, 25, 50 and 100 mg/mL by proportional dilution of the primary stock. The secondary stock solutions were mixed by inversion, and ranged in
appearance from clear and colorless to clear and light yellow, with no evidence of precipitates.

To prepare a batch of sediment for each treatment level, a 18-mL volume of the appropriate stock solution was added to 90 grams of sand in a
labeled glass beaker and was stirred with a glass stir rod until homogenous. This dosed “sand premix” was placed under a fume hood and the
acetone was allowed to evaporate for approximately one hour. The 90 gram sand premix was added to 810 grams of untreated formulated sediment
in a 2000 mL plastic Nalgene® bottle and mixed on a rotary mixer for approximately one-half hour. Additional formulated sediment (900 g) was
added to the premix to achieve a final weight of 1800 grams. This 1800-g batch sediment was mixed on a rotary mixer for approximately 16 hours
prior to transfer of the dry sediment to the test compartments. Since a solvent (acetone) was used in the preparation of the test sediments, a solvent control was included in the test design. The solvent control sediment was prepared using 18 mL of acetone, with the same mixing procedures as the
treated sediments but with no test substance added. The negative control sediment was prepared without the addition of test substance or solvent.
Test organisms (species):
Chironomus riparius
Details on test organisms:
The midge, Chironomus riparius, was selected as the test species for this study. This species is representative of an important group of aquatic invertebrates and was selected for use in the study based upon past history of use in the laboratory. Midges used in the test were obtained as egg masses from Environmental Consulting and Testing, Superior, Wisconsin. The identity of the species was verified by the supplier.

Larvae used to start the test were collected from six separate egg masses and were hatched in water from the same source and at approximately the same temperature as was used during the test. At the time of test initiation, the larvae were 1 to 4 days old. During the holding period, water temperatures ranged from 19.0 to 19.5¿C, the pH of the water ranged from 8.4 to 8.8, and the dissolved oxygen concentrations were >9.1 mg/L (100% of saturation). The larvae showed no signs of disease or stress prior to test initiation.
Study type:
laboratory study
Test type:
static
Water media type:
freshwater
Type of sediment:
artificial sediment
Limit test:
no
Duration:
28 d
Exposure phase:
total exposure duration
Post exposure observation period:
None
Hardness:
Hardness (mg/L as CaCO3) - Control 140, Solvent control - 148, Treated - 148
Test temperature:
19.4– 20.6°C
pH:
8.2 - 9.2
Dissolved oxygen:
=82%; aerated
Salinity:
N/A
Ammonia:
< LOQ(< 0.17 mg/L as NH3)
Nominal and measured concentrations:
Nominal: 63, 125, 250, 500, 1000 mg/kg dry sediment

Mean Measured: 41, 94, 178, 388, 1048 mg/kg


Details on test conditions:
Test Apparatus
The test chambers were 1-quart glass jars containing approximately 2 cm (approximately 130 g) of sediment and approximately 600 mL of overlying water. The depth of the sediment measured in a representative chamber was 1.7 cm, and the depth of the overlying water in a representative compartment was 8.7 cm. The approximate 1:4 ratio of sediment depth to overlying water depth was maintained throughout the test by periodically replacing water lost due to evaporation with reverse-osmosis water. Loose plastic covers were placed over each test chamber during the test, and aeration was applied to each test chamber through a glass pipette that extended to a depth not closer than 2 cm to the surface of the sediment. Air was bubbled into the test chambers at rate greater than 1 bubble per second, a rate that did not disturb the sediment. The test chambers were indiscriminately arranged in a temperature-controlled environmental chamber during the test, and were labeled with the project number, test concentration and replicate designation. The general operation of the test apparatus was checked visually at least once each day during the test.
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
299 mg/kg sediment dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
emergence rate
Remarks on result:
other: 254-354, 95%CI
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
94 mg/kg sediment dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
emergence rate
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
178 mg/kg sediment dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
emergence rate
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
388 mg/kg sediment dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
development rate
Details on results:
Measurement of Test Concentrations
Nominal concentrations selected for use in this study were 63, 125, 250, 500 and 1000 mg/kg dry weight of sediment. During the course of the test, the appearance of the overlying water was observed in the test chambers. At test initiation and termination, the overlying water appeared ranged in appearance from cloudy and yellow to clear and yellow.

Results of analyses to measure the concentration of TPS 44 in the stock solution samples used to dose the sediment are presented in the report. Measured concentrations ranged from approximately 87 to 107% of nominal. Results of analyses to measure concentrations in the sediment, overlying water and pore water samples during the test are presented in the report. All reported sediment concentrations are expressed on a dry weight basis with an LOQ of 10.0 mg/kg dry sediment. The LOQ for overlying and pore water analyses was 0.00250 mg/L. Measured concentrations of the test substance in negative and solvent control sediment and water samples on Days 0, 7 and 28 were below the LOQ. Measured concentrations in the sediment samples collected from the treatment groups ranged from approximately 59 to 117% of nominal. Measured concentrations in the overlying water and pore water samples ranged from approximately
The percentage of TPS 44 in the sediment, overlying water and pore water based on mass balance calculations of measured concentrations are summarized in Table 5, and mass balance calculations at each sampling interval are presented in Appendix 9. Mass balance of the test substance indicates the concentration in the total test system remained fairly consistent. TPS 44 remained mostly in the sediment during the exposure period. At study start approximately 81.7% of the total amount of TPS 44 was in the sediment, and by the end of the study the amount in the sediment decreased to 76.2%. Only a negligible percentage of material was measured in the overlying water and pore water during the study.

Observations and Measurements
Measurements of temperature, dissolved oxygen and pH of the overlying water in the test chambers are summarized in Table 6, and individual measurements are presented in Appendices 10, 11 and 12, respectively. All water quality measurements were within the desired ranges. Water temperatures were within the 20 ± 2°C range established for the test. Measurements of pH ranged from 8.2 to 9.2 during the test. Dissolved oxygen concentrations remained =7.4 mg/L (=82% of saturation) throughout the test.. Measurements of specific conductance, hardness, alkalinity and ammonia of the overlying water in the negative control and the highest concentration treatment group are summarized in Table 6 and individual measurements are presented in Appendix 13. The Day 0 measurements were consistent with typical water quality of Wildlife International well water, while the Day 28 measurements were higher than typical water quality of Wildlife International well water (Appendix 3). The higher measurements at the end of the test may be a result of the static nature of the test. The departure from typical measurements did not have a negative impact on the results of the test as is demonstrated by the control groups meeting all validity criteria established for the test by the OECD 218 guideline (1). Measurements of ammonia in the overlying water at the beginning and end of the test were below the limit of detection (LOD of <0.17 mg/L, the lowest calibration standard). Light intensity at test initiation was 575 lux at the surface of the water of one representative test chamber.

No unusual observations of organisms avoiding the sediment occurred during the test. There were a few observations of larvae on the surface of the sediment, climbing the walls of the test chamber, or swimming in the water column during the study, but these did not appear to be dose-responsive, were comparable between the control and treatment groups, and were not considered to be treatment-related. There also were a few observations of dead pupae or larvae and adults that emerged and died. However, these numbers were small, were not concentration-responsive, and were not considered to be treatment-related.

There was an apparent treatment-related effect on emergence in the 178, 388 amd 1048 mg/kg treatment groups. The numbers of emerged midges and emergence ratios in each control and treatment group are summarized in Table 7, and daily counts of emergence in individual replicates during the period of emergence are presented in Appendix 14, with replicate totals and emergence ratios presented in Appendix 15. Emergence was first noted on Day 13 of the test and continued through Day 22. The mean emergence ratios in the negative control, solvent control and the 41, 94, 178, 388 and 1048 mg/kg treatment groups were 0.94, 0.90, 0.90, 0.81, 0.63, 0.33 and 0.15, respectively. The 28-day EC50 for emergence was determined to be 299 mg/kg, with a 95% confidence interval of 254 to 354 mg/kg. Dunnett’s test indicated that there was a statistically significant difference in emergence in the 178, 388 and 1048 mg/kg treatment groups in comparison to the pooled control (p = 0.05). Therefore, the LOEC for emergence ratio was determined to be 178 mg/kg and the NOEC was 94 mg/kg.

There was an apparent treatment-related effect on development rate (defined as the portion of larval development which takes place per day) in the 1048 mg/kg treatment groups. The mean development times and development rates in each control and treatment group are summarized in Table 7, and individual replicate data are presented in Appendix 16. The mean development times in the negative control, solvent control and the 41, 94, 178, 388 and 1048 mg/kg treatment groups were 15.2, 15.2, 15.1, 15.3, 15.5, 15.8 and 17.5, respectively, and the mean development rates were 0.0685, 0.0689, 0.0693, 0.0684, 0.0672, 0.0657 and 0.0593, respectively. Dunnett’s test indicated that there was no statistically significant difference in development time (p > 0.05), but there was a statistically significant difference in development rate in the 1048 mg/kg treatment groups in comparison to the pooled control (p = 0.05). Therefore, the LOEC for development rate was determined to be 1048 mg/kg and the NOEC was 388 mg/kg.
Reported statistics and error estimates:
The 28-day EC50 for emergence was determined to be 299 mg/kg, with a 95% confidence interval of 254 to 354 mg/kg. Dunnett’s test indicated that there was a statistically significant difference in emergence in the 178, 388 and 1048 mg/kg treatment groups in comparison to the pooled control (p = 0.05). Therefore, the LOEC for emergence ratio was determined to be 178 mg/kg and the NOEC was 94 mg/kg.

There was an apparent treatment-related effect on development rate (defined as the portion of larval development which takes place per day) in the 1048 mg/kg treatment groups. The mean development times in the negative control, solvent control and the 41, 94, 178, 388 and 1048 mg/kg treatment groups were 15.2, 15.2, 15.1, 15.3, 15.5, 15.8 and 17.5, respectively, and the mean development rates were 0.0685, 0.0689, 0.0693, 0.0684, 0.0672, 0.0657 and 0.0593, respectively. Dunnett’s test indicated that there was no statistically significant difference in development time (p > 0.05), but there was a statistically significant difference in development rate in the 1048 mg/kg treatment groups in comparison to the pooled control (p = 0.05). Therefore, the LOEC for development rate was determined to be 1048 mg/kg and the NOEC was 388 mg/kg.

 

Summary of Emergence and Development of Midges (Chironomus riparius)

During the 28-Day Exposure to Sediment-Incorporated TPS 44

Mean Measured

Sediment

Concentration

(mg/kg)

Number Emerged1

 

Mean

Emergence

Ratio2,4

Mean

Development

Time

(Days)4

 

Mean

Development

Rate3,4

Males

Females

Total

Negative Control

43

32

75

0.94(± 0.09)

15.2(± 0.51)

0.0685(± 0.0022)

Solvent Control

42

30

72

0.90(± 0.08)

15.2(± 0.63)

0.0689(± 0.0029)

Pooled Control

85

62

147

0.92(± 0.08)

15.1(± 0.53)

0.0687(± 0.0024)

41

42

30

72

0.90(± 0.04)

15.1(± 0.12)

0.0693(± 0.0007)

94

37

28

65

0.81(± 0.06)

15.3(± 0.66)

0.0684(± 0.0028)

178

36

14

50

 0.63(± 0.10)*

15.5(± 0.38)

0.0672(± 0.0017)

388

12

14

26

 0.33(± 0.09)*

15.8(± 0.92)

0.0657(± 0.0039)

1048

7

5

12

 0.15(± 0.13)*

 17.5(± 1.54)

 0.0593(± 0.0049)*

1 Each replicate contained 20 midge larvae at test initiation, for a total of 80 larvae per control and treatment group.

2 Emergence ratio is calculated as the number of emerged midges divided by the initial number exposed, and corresponds to percent emergence.

3 The development rate represents that portion of larval development which takes place per day.

4 Calculated using Excel 2010. Manual calculations may differ slightly.

*Indicates a statistically significant difference in comparison to the pooled control (p = 0.05) using Dunnett’s test.

28-Day EC50for emergence: 299 mg/kg, with a 95% confidence interval of 254 to 354 mg/kg.

Validity criteria fulfilled:
yes
Conclusions:
Midges (Chironomus riparius) were exposed for 28 days to five mean measured concentrations of sediment-incorporated TPS 44 ranging from 41 to 1048 mg/kg. There were treatment-related effects observed on emergence at 178, 388 and 1048 mg/kg and on development time and development rate at 1048 mg/kg. Based on the mean measured concentrations in sediment, the 28 day EC50 value for emergence was 299 mg/kg, with a 95% confidence interval of 254 to 354 mg/kg. The slope of the concentration-response curve was 2.0. Based on the effects observed on emergence ratio, the LOEC for the study was 178 mg/kg and the NOEC was 94 mg/kg.
Executive summary:

SUMMARY

 

 

SPONSOR:

Arkema

 

 

STUDY TITLE:

TPS 44: A Prolonged Sediment Toxicity Test with the Midge (Chironomus riparius) Using Spiked Sediment

 

 

WILDLIFE INTERNATIONAL PROJECT NUMBER:

524A-128

 

 

GUIDELINE:

OECD 218

 

TEST SUBSTANCE:

Name:

TPS 44

(Polysulfure de tertiobutyle)

 

Purity (Content of A.I.):

93.8%

 

Batch/Lot Number:

02-april-2013

 

Appearance:

Liquid

 

 

 

TEST DATES:

Experimental Start (OECD):

June 6, 2014

 

Experimental Start (EPA):

June 11, 2014

 

Exposure Termination:

July 9, 2014

 

Experimental Termination:

July 12, 2014

 

 

 

LENGTH OF EXPOSURE:

28 Days under static conditions

 

TEST ORGANISM:

Name:

Midge (Chironomus riparius)

 

Source:

Environmental Consulting and Testing

 

 

Superior, Wisconsin 54880

 

Age:

First-instar larvae, 1-4 Days at test initiation

 

 

REPLICATION:

4 test chambers per test concentration, 20 midge larvae per chamber (total of 80 midges per concentration)

 

ENVIRONMENTAL CONDITIONS IN OVERLYING WATER:

 

Temperature Range:

 

19.4– 20.6°C

 

Dissolved Oxygen:

=82%; aerated

 

pH Range:

8.2 – 9.2

 

Hardness Range:

140 – 316mg/L as CaCO3

 

Alkalinity Range:

178 – 320mg/L as CaCO3

 

Specific Conductance Range:

401 – 627µS/cm

 

Ammonia:

< LOQ(< 0.17 mg/L as NH3)

TEST CONCENTRATIONS IN SEDIMENT:

 

 

Nominal

Mean Measured

 

(mg/kg dry sediment)

(mg/kg)

 

Negative Control

< LOQ

 

Solvent Control

< LOQ

 

63

41

 

125

94

 

250

178

 

500

388

 

1000

1048

 

MEASURED ENDPOINTS:

Total number of midges emerged and development time; used to calculate emergence ratio and development rate

 

RESULTS: 

Based on mean measured concentrations in sediment:

 

 

28-Day EC50 for Emergence:

299 mg/kg

 

95% Confidence Interval:

254 - 354 mg/kg

 

 

 

 

The most sensitive parameter was emergence

 

Lowest-Observed-Effect Concentration:

178 mg/kg

 

No-Observed-Effect Concentration:

94 mg/kg

Description of key information

A study about chronic toxicity of polysulfides, di-tert-butyl to freshwater sediment organisms has been carried out, according to OECD TG 218. The NOEC for emergence rate of chironomids sets at 94 mg/kg dw.

Key value for chemical safety assessment

EC50 or LC50 for freshwater sediment:
299 mg/kg sediment dw
EC10, LC10 or NOEC for freshwater sediment:
94 mg/kg sediment dw

Additional information

According to physico-chemical properties of polysulfides, di-tert-butyl the target compartment, in case of release to water, would be sediment. Consequently, a study of possible impacts on sediment organisms has been proposed in the frame of preparation of the REACH registration dossier. Further to the final decision of ECHA agreeing with the registrant testing proposal, a study according to OECD TG 218 has been carried out: a prolonged sediment toxicicty test with the midge Chironomus riparius using spiked sediment.

The results are summarised in the robust study summary in the accompanying dataset. The validity criteria are fulfilled and a neat concentration effect relationship has been observed. The most sensitive endpoint is the emergency rate, EC50 and NOEC are provided according to this parameter, and set at: EC50 = 299 mg/kg dw, NOEC = 94 mg/kg dw. No significant difference between males and females have been observed. Exposure has been done using spiked sediment. Monitoring of concentrations has been done at t = 0, 7 and 28 days. Measured concentrations in the sediment samples collected from the treatment groups renged from 59 to 117 %. Measured concentrations in the overlying and pore water samples ranged from < LOQ (0.0025 mg/L) to < 1% of nominal, indicating that test substance in the overlying and pore water samples was negligible, and indeed that the sediment is the sink of the polysulfide, di-tert butyl. Results of the study were based on mean measured concentrations in sediment.

Besides, the NOEC obtained in this study can be used to derive a NOECaqua making use of the Equilibrium Partitioning Method, as described in Guidance, R.11.1.3.3. This NOECaqua can be used the "T" criterion in PBT assessment.