Bis(dibutyldithiocarbamato-S,S')copper

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:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Deviation: an experimental error occurred when the biomasses of both control groups (solvent and solvent-free) were determined. These biomasses were abnormal and deemed invalid by the study director. Instead, the biomasses produced in the treated groups were compared to historical control data from the CRO and from the ring-test used to develop OECD 225 TG.

Qualifier:

according to guideline

Guideline:

OECD Guideline 225 (Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment)

Deviations:

yes

Remarks:

An error occurred with the determination of the final biomass of both control groups. The treated groups were compared to historical control data from the CRO and to the data from the ring-test used to develop OECD 225 TG

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

None.

Analytical monitoring:

yes

Details on sampling:

An additional test vessel was prepared for the control, solvent control and each test concentration for use as ‘destructive’ sample for the analysis of water, sediment and pore water concentrations on Day 0 (worm addition) and Day 28. The destructive sampling vessels were prepared in the same manner as the test vessels. The protocol stated that worms would be added to the destructive test vessels however, the destructive vessels were prepared without the addition of worms. This was considered not to impact on the integrity of the test given that the vessels were maintained under the same test conditions and were used to indicate the concentration of the test substance in the sediment/water on Day 0 and after 28 days under test conditions.Samples of sediment from the control, solvent control and each test concentration were taken for analysis on Day -2 (day of sediment preparation). Samples of the overlying water, sediment and pore water from the destructive test vessels on Day 0 (day of organism addition) and Day 28 were taken for analysis from the control, solvent control and each test concentration.

Vehicle:

yes

Details on sediment and application:

Test Substrate - Artificial Sediment
The artificial sediment used for the test was prepared in accordance with OECD Guideline 225. On the basis of dry weights, the constituents were as follows:
• 4-5% (dry weight) peat
• 20% (dry weight) kaolin clay
• 75-76% (dry weight) quartz sand
• 0.4-0.5% (dry weight) powdered stinging nettle leaves
To minimise the possibility of elevated ammonia concentrations at the start of the test, the peat was pre-conditioned prior to use. A suspension of the required amount of peat was prepared in reverse osmosis water using a high performance homogenising device and the pH adjusted to 5.5 ± 0.5 with CaCO3. The suspension was then conditioned with gentle stirring to stabilise pH and establish a stable microbial content for three days. The required amount of peat suspension was then mixed with the required amount of sand and clay to obtain homogenous sediment with a water content of 30% to 50% dry weight of the sediment. The final pH of the sediment was adjusted to 7.0 ± 0.5 by the addition of calcium carbonate where required. The sediment was then pre-conditioned, under test conditions including overlying water, for seven days. Prior to addition of the test substance, the overlying water was siphoned from the tank. The organic carbon content of the prepared sediment was 1.8% which was within the required range stated in the protocol of 2 ± 0.5%.

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

The test species used for the study was Lumbriculus variegatus. The strain used was from in-house cultures that were obtained from Aquatic Research Organism, New Hampshire, USA (originally supplied by United States Fish and Wildlife Service, Missouri, USA). Due to the reproduction mode of Lumbriculus variegates (architomy or morphallaxis), age synchronisation of the worms was conducted prior to testing to minimise uncontrolled reproduction and regeneration, which may have resulted in high variation in test results. Fourteen days prior to the start of exposure, large (adult) worms were artificially fragmented by dissecting in the median body region with a scalpel. The posterior ends were placed in a culture vessel, containing shredded paper towel with overlying dechlorinated mains water, and left to regenerate. The worms were held using flow-through conditions under the same conditions as the main culture. The temperature at which the worms were maintained could not be confirmed from the data. This was a deviation to the protocol which stated that the worms would be maintained at a temperature of 20 ± 2ºC. This deviation was considered not to affect the integrity of the test as the worms were healthy when added to the test vessels. The worms were fed from seven days after dissection onwards with 2-5 mL of a 100 mg/mL suspension of Tetramin®.

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:

100 - 230 mg/L as CaCO3 (min-max)

Test temperature:

18.6 - 20.7 °C (min-max)

pH:

7.46 - 8.83 (min-max)

Dissolved oxygen:

41 - 100% saturation (min-max)

Salinity:

Freshwater.

Ammonia:

1 - 5.4 mg/L N-NH3 (overall mean at D0 - overall mean at D28)

Conductivity:

Not measured.

Nominal and measured concentrations:

Nominal: control ; solvent control ; 10 ; 32 ; 100 ; 320 ; 1000 mg/kg sediment dw
Measured: see Table 1

Details on test conditions:

A definitive test was conducted at nominal test substance concentrations of 10, 32, 100, 320 and 1000 mg/kg (dry weight). Control and solvent control groups were also included. Age synchronised Lumbriculus were exposed to the test or control conditions for a 28-day period, without renewal of the test medium. Four replicates were prepared for the 10, 32, 100 and 320 mg/kg test concentrations along with six replicates for the control, solvent control and 1000 mg/kg test concentration. The study was conducted in glass beakers (250 mL) containing a layer of sediment and water to give a sediment to water depth ratio of 1:4 (sediment added to the 50 mL mark with overlying water added to the 250 mL mark). The water level was marked on each vessel, so evaporative losses could be replaced with deionised water during the test.
A concentrated solvent stock solution was prepared by weighing ca 2000 mg of test substance and dissolving in 20 mL of acetone resulting in an initial 100 mg/mL solvent stock solution. Serial dilutions were prepared in acetone for the remaining solvent stock solutions of 32, 10, 3.2 and 1.0 mg/mL. Each concentration was prepared as detailed in table 2.
Following addition of the test substance, each bulk sediment was carefully and thoroughly mixed by hand. The bulk sediment was then distributed between the appropriate test vessels and overlying water added. To avoid separation of the sediment and to reduce turbidity of the test water, the sediment was covered with a plastic disc whilst vessels were filled with treated mains water. This disc was removed after filling.
After approximately 72 hours of acclimation, 10 age-synchronised Lumbriculus were added to each vessel. Following the addition of the test organisms to the test vessels, aeration was provided through a narrow bore glass tube. All vessels were covered with a clear plastic lid. An additional test vessel was prepared for the control, solvent control and each test concentration for use as ‘destructive’ sample for the analysis of water, sediment and pore water concentrations on Day 0 (worm addition) and Day 28. The destructive sampling vessels were prepared in the same manner as the test vessels. The protocol stated that worms would be added to the destructive test vessels however, the destructive vessels were prepared without the addition of worms. This was considered not to impact on the integrity of the test given that the vessels were maintained under the same test conditions and were used to indicate the concentration of the test substance in the sediment/water on Day 0 and after 28 days under test conditions.

Feeding
The food source (powdered stinging nettle leaves) was added to the sediment prior to spiking with the test substance. No additional food was supplied during the test.

Observations and Water Quality Determinations
Test vessels were observed daily to visually assess any behavioural differences in worms compared to the controls, for example, sediment avoidance, faecaland faecal pellets visible on the sediment surface. At the end of the test, the number of live and dead worms per replicate were counted and recorded. Once removed, the live worms were transferred to pre-weighed and labelled vessels (one per replicate). The vessels were placed in an oven at 105 ºC overnight and the dry weight per replicate determined.
Test cultures were maintained in a temperature-controlled laboratory, nominally at 20 ±2 ºC. Day length was controlled to give a photoperiod of 16 hours light:8 hours darkness. The light intensity was maintained at 100 to 500 lux. The pH, temperature and dissolved oxygen were recorded in one replicate test vessel for the controls and each test concentration on Days 0 and 28 and weekly throughout the duration of the test. The total water hardness was measured, using a pooled sample, in the controls and each test concentration on Days 0 and 28. Total ammonia concentration was measured, using a pooled sample, in the controls and each test concentration on Day 0 and three times a week for the duration of the test. Light intensity of the test area was also measured at the start and end of the test.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

320 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

total number of worms

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

Reproduction
The numbers of Lumbriculus variegatus in each individual test vessel, along with the means per treatment level, are presented in Table 3. No statistically significant effect (p=0.05) was observed on the numbers of worms for the 10 to 320 mg/kg test concentrations compared to the controls (which were pooled because no significant difference was observed between them). A statistically significant (p<0.05) decrease in the numbers of worms was observed at the 1000 mg/kg test concentration compared to the controls.
The EC50 value for reproduction was >1000 mg/kg (dry weight). The NOEC and LOEC for reproduction were determined to be 320 and 1000 mg/kg (dry weight), respectively.

Biomass
Observations indicate that while the test concentrations resulted in comparable worm biomasses, the control and solvent control were significantly higher than expected. Potential causes of this could be procedures related to the cleaning, drying and weighing for the control and solvent control treatment levels. It was possible that:

1. Excess water was not dried off completely from the worms at the time of weighing.

2. The worms were not sufficiently cleaned before the drying phase was initiated and that some sediment residue was carried over with the worms.

However, it is not possible to confirm the direct cause in the discrepancy between the control and solvent control weights with the treatment levels. Since the biomasses of the controls are unreliable, the biomass of the treated groups were not compared to them. Instead, statistical analysis was performed using the Bonferroni Adjusted t Test and the Tukey-Kramer Test to compare the five treatment levels for significant differences between treatments. A comparison of each treatment level to the lowest treatment tested was also performed and showed no significant differences. Both the Bonferroni Adjusted t Test and the Tukey-Kramer Test indicated that no statistically significant differences were observed in the definitive test between the treatment levels, therefore, the data is considered scientifically robust and indicated that no significant test substance related effect were observed in the treatments during the test.
When compared to the weights observed in the test concentrations, historical control data show comparable weights indicating that the treatment levels performed as expected and showed no test substance related effects and remained within the historical control range.
Data from the OECD 225 Lumbriculus ring test indicated that the number of worms and total biomass generally correlated with a mean inter-laboratory worm weight of 0.025 g in the control treatment and 0.027 g in the solvent control. This was comparable to the weights observed in the definitive test treatment levels.
Based on the comparisons with the historical control and solvent control data and ring test data, the NOEC for biomass was estimated to be 1000 mg/kg (dry weight), without any LOEC. In other words, the test substances did not have any effect on worm biomass during the study at any tested concentration.

Results with reference substance (positive control):

None.

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v 1.8.6.8. The EC50 is defined as the concentration that results in a 50% mean reduction, respectively, relative to the control. The No Observed Effect Concentration (NOEC) is defined as the concentration which caused no statistically significant (p=0.05) reduction compared to the control. The Lowest Observed Effect Concentration (LOEC) is defined as the concentration immediately above the NOEC.

Worm number
Data from the control and solvent control groups were compared using the Equal Variance t Two-Sample test. No significant difference (p=0.05) was observed between the control and solvent control groups in terms of reproduction (worm numbers)
The NOEC and LOEC for reproduction were determined using the Bonferroni Adjusted t Test. EC50 values, and where applicable the EC10 and EC20 values, were determined using linear regression.

Biomass
Due to a technical error during the study, no statistical comparison could be made with any control group. A weight of evidence approach including historical control data showed that the test substance did not have any effect on biomass (see the result section for more details).

Table 1: Measured concentrations of test substance within the exposure system following application to sediment

Nominal concentration (mg/kg)	Concentration in overlying water (mg./L)	Concentration in sediment (mg/kg)	% of nominal concentration in sediment	Mean % of nominal concentration in sediment
Day -3
Control	-	<LOQ <LOQ	- -	-
Solvent control	-	<LOQ <LOQ	- -	-
10	-	9.54 10.1	95 101	98
32	-	19.9 21.5	62 67	65
100	-	92.0 66.2	92 66	79
320	-	144 129	45 40	43
1000	-	683 735	68 74	71
Day 0
Control	<LOQ <LOQ	<LOQ <LOQ	- -	-
Solvent control	<LOQ <LOQ	<LOQ <LOQ	- -	-
10	<LOQ <LOQ	6.10 6.40	61 64	63
32	<LOQ <LOQ	17.4 24.1	54 75	65
100	<LOQ <LOQ	63.4 63.8	63 64	64
320	<LOQ <LOQ	104 198	64 62	63
1000	<LOQ <LOQ	808 737	81 74	77
Day 28
Control	<LOQ <LOQ	<LOQ <LOQ	- -	-
Solvent control	<LOQ <LOQ	<LOQ <LOQ	- -	-
10	<LOQ <LOQ	3.74 3.41	37 34	36
32	<LOQ <LOQ	16.8 14.8	52 46	49
100	<LOQ <LOQ	42.9 47.3	43 47	45
320	<LOQ <LOQ	188 177	59 55	57
1000	<LOQ <LOQ	796 610	80 61	70
- Not applicable LOQ – Limit of Quantification of the analytical method (assessed as 0.01 mg/L for aqueous media and 1.0 mg/kg for sediment)

Table 2: Preparation of the test substance prior to application to sediment.

Concentration (mg/kg)	Concentration of stock solution (mg/mL)	Volume of stock solution added (mL)	Weight of sand dosed (g)	Weight of prepared sediment (g)	Final weight of sediment prepared (g)
Control	–	–	90	810	900
Solvent control	acetone	9	90	810	900
10	1.0	7	70	630	700
32	3.2	7	70	630	700
100	10	7	70	630	700
320	32	7	70	630	700
1000	100	9	90	810	900

Sediment weights are dry weight of sediment

Table 3: numbers of Lumbriculus variegatus per treatment at the end of the test.

Nominal concentration (mg/kg)	Mean number of worms on Day 28
Control	32
Solvent control	36
10	30
32	33
100	32
320	30
1000	29*

                                 * Significant difference (p<0.05) compared to the solvent control

Table 4: dry weights of live Lumbriculus variegatus per treatment at the end of the test.

Nominal concentration (mg/kg)	Mean dry weight per worm (mg)
Control	3.82 (unreliable due to technical error)
Solvent control	2.75 (unreliable due to technical error)
10	1.36
32	1.35
100^1	1.85
320	1.29
1000	1.19
^1A single replicate was excluded from the mean and statistical analysis given that a value of zero was recorded for the worm weights. This was considered to be erroneous given the number of worms present in the replicate.

Validity criteria fulfilled:

yes

Conclusions:

NOEC for reproduction was 320 mg/kg substance dw. No significant effect was observed on biomass.

Executive summary:

The objective of the study was to assess the effects of the test substance on the reproduction and biomass of the endobenthic aquatic oligochaete Lumbriculus variegatus by exposure in a static sediment - water exposure system over a 28-day period. The study was conducted in accordance with the requirements of OECD Chemicals Testing Guideline No.225: Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment (adopted 16 October 2007).

Based on the results of the range-finding test, which is not fully reported, the definitive test was conducted at nominal test concentrations of 10, 32, 100, 320 and 1000 mg/kg (dry weight). Control and solvent control groups were also included. Four replicates of 10 age-synchronised worms per replicate were prepared for the 10, 32, 100 and 320 mg/kg test concentrations, along with six replicates for the control, solvent control and 1000 mg/kg test concentration, and exposed for 28 days.

Analysis of the sediment on Day -3 (day of preparation) resulted in measured concentrations of 98, 65, 79, 43 and 71% of nominal value for the 10, 32, 100, 320 and 1000 mg/kg test concentrations, respectively.

Analysis of the Day 0 (day of organism addition) sediment samples yielded measured concentrations of 63, 65, 64, 63 and 77% of nominal value for the 10, 32, 100, 320 and 1000 mg/kg test concentrations, respectively. Measured concentrations of the overlying water samples were less than the LOQ of the analytical method.

Analysis of the Day 28 samples yielded measured concentrations of 36, 49, 45, 57 and 70% of nominal value for the 10, 32, 100, 320 and 1000 mg/kg test concentrations, respectively, with no detectable concentrations above the LOQ in overlying water.

Measured concentrations in the sediment were, in general, outside the 80% to 120% of nominal acceptance criteria. The Day 0 data indicated that the exposure system had been correctly dosed since analytical recoveries were consistent and followed the expected concentration gradient. The lower Day 28 recoveries were therefore possibly due to irreversible binding of the test substance to the sediment or possible degradation of the test substance which could not be detected with the analytical method.

Given that no detectable concentrations above the LOQ were observed in the overlying water, it was considered that the test substance remained in the sediment layer throughout the 28-day test period with little partitioning to the overlying water layer.

No test substance was detected above the LOQ in the controls during the test.

These results indicated that the test substance remained in the sediment layer throughout the 28-day test period with little partitioning to the overlying water layer.

Significant effect (p<0.05) on reproduction were observed during the test compared to controls. The NOEC value for reproduction was 320 mg/kg dw. No significant effect was observed on biomass at any tested concentration.

Description of key information

The objective of the study was to assess the effects of the test substance on the reproduction and biomass of the endobenthic aquatic oligochaete Lumbriculus variegatus by exposure in a static sediment - water exposure system over a 28-day period. The study was conducted in accordance with the requirements of OECD Chemicals Testing Guideline No.225: Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment (adopted 16 October 2007).

The test was conducted at nominal test concentrations of 10, 32, 100, 320 and 1000 mg/kg (dry weight). Control and solvent control groups were also included. Four replicates of 10 age-synchronised worms per replicate were prepared for the 10, 32, 100 and 320 mg/kg test concentrations, along with six replicates for the control, solvent control and 1000 mg/kg test concentration, and exposed for 28 days.

Analysis of the sediment on Day -3 (day of preparation) resulted in measured concentrations of 98, 65, 79, 43 and 71% of nominal value for the 10, 32, 100, 320 and 1000 mg/kg test concentrations, respectively. Analysis of the Day 0 (day of organism addition) sediment samples yielded measured concentrations of 63, 65, 64, 63 and 77% of nominal value for the 10, 32, 100, 320 and 1000 mg/kg test concentrations, respectively. Measured concentrations of the overlying water samples were less than the LOQ of the analytical method. Analysis of the Day 28 samples yielded measured concentrations of 36, 49, 45, 57 and 70% of nominal value for the 10, 32, 100, 320 and 1000 mg/kg test concentrations, respectively, with no detectable concentrations above the LOQ in overlying water. These results indicated that the test substance remained in the sediment layer throughout the 28-day test period with little partitioning to the overlying water layer.

Significant effect (p<0.05) on reproduction were observed during the test compared to the controls. The NOEC value for reproduction was 320 mg/kg dw. No significant effect was observed on biomass at any tested concentration.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

320 mg/kg sediment dw