Sodium dimethyldithiocarbamate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Jan - 08 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM E 1193-97 Standard Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and every treatment group
- Sampling method: Duplicate water samples were collected from alternating replicate test chambers in each treatment and control group at the beginning of the test, at approximately weekly intervals during the test and at the end of the test. At each interval, one set of samples was processesed immediately for analysis, while the second set of samples was stabilized and stored frozen as backup for potential future analysis. All samples were collected volumetrically from mid-depth (20.0 mL for negative control and 10.0 mL for the treatment groups), placed in glass vials to which iso-octane was added to stabilize the sample (3.0 mL for the negative control and 10.0 mL for the treatment groups). The vial was capped after the solvent iso-octane was added to each sample and the samples were processed immediately for analysis or stored frozen until analysis.
- Sample storage conditions before analysis: Frozen

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Individual stock solutions were prepared for each of the five concentrations tested and were prepared five times during the study. A primary stock solution with a concentration of 1800 µg a.i./mL was prepared in reverse osmosis (RO) water. The primary stock solution was mixed by 15 min sonication, followed by inversion. The stock solution was clear and colorless, without precipitation. Four secondary stock solutions with nominal concentrations of 46, 116, 280, and 720 µg a.i./mL were prepared by dilution of the primary stock in RO water. The secondary stock solutions were mixed by inversion, and appeared clear and colorless with no precipitates. The stock solutions were stored refrigerated in glass volumetric flasks or glass amber bottles with Teflon-lined lids and aliquots of each stock were placed in a syringe every 2 - 4 d during the study.
- Differential loading: No
- Controls: yes, dilution water only
- Evidence of undissolved material: No, the stock solutions appeard clear and colorless with no visible precipitate.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea (Cladoceran)
- Source: In-house cultures maintained by the testing facility
- Age of neonates used in the test: < 24 h neonates
- Culture conditions: Adult daphnids were cultured in water from the same source and at approximately the same conditions as in the test (19.3 - 20.6 °C, pH 8.1 - 8.5 and ≥ 7.9 mg/L oxygen (≥ 87% of saturation).
- Feeding during test: Yes
- Food type: A mixture of yeast, cereal grass media and trout chow (YCT) supplemented with a suspension of the freshwater green alga Raphidocelis subcapitata and a vitamin stock solution.
- Amount: 0.8 mL of YCT and 1.5 mL of algae per feeding (corresponding to 0.4 - 0.7 mg C/daphnid/d). In addition, each test chamber also received 0.5 mL of vitamine solution once daily. An excess amount was fed in order to maintain sufficient feed in the flow-through system to support acceptable reproduction rates (recommended amount: 0.1 - 0.2 mg C/daphnid/d).
- Frequency: Daphnids were fed two or three times per day though Day 7 of the test and then were fed three to four times per day until the last day of the test.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Three adult daphnids were used to supply neonates for the test. These were held 14 d prior to the collection of juveniles for testing and had each produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 8-day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

128 - 144 mg/L as CaCO3

Test temperature:

20 ± 1 °C

pH:

7.7 - 8.6

Dissolved oxygen:

8.8 - 9.1 mg/L
(≥ 98% of saturation)

Conductivity:

317 - 348 µS/cm

Nominal and measured concentrations:

Control, 2.3, 5.8, 14, 36, and 90 µg a.i./L (nominal)
< LOQ, 1.0, 3.4, 9.5, 26, and 78 µg a.i./L (arithmetic mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: 7 L glass aquaria filled with approximately 6 L of test medium. The volume in the test chambers was maintained by an overflow port on the side of the test chamber. The daphnids were held in test compartments suspended in each of four test chambers and covered with plexiglass. The test compartments were 300 mL glass beakers, approximately 6.5 cm in diameter and 12 cm in height, containing approximately 225 mL test solution. Nylon mesh screens covered two holes on opposite sides of each test compartment to permit test solution to flow in an out of the compartment. The depth of the test water in a representative compartment was approximately 8 cm, while the depth of water in a representative test chamber was approximately 16 cm.
- Type of flow-through: The toxicity test was conducted with an exposure system consisting of a continuous-flow diluter used to deliver each concentration of the test item and a negative control (dilution water) to the test vessels. Syringe pumps were used to deliver the test item stock solutions to randomly assigned mixing chambers where the stocks were mixed with dilution water prior to delivery to the test chambers. The stock solution was diluted with UV sterilized well water in the mixing vessels in order to obtain the desired test concentrations prior to delivery to the test vessels. After mixing, the test solution in each mixing chamber was pumped into the appropriate replicate test chamber using a persistaltic pump.
- Renewal rate of test solution: The flow of dilution water into each mixing chamber was controlled with rotameters and was adjusted to provide approximately 8 volume additions of test water in each test chamber per day. It was ensured that flow rates did not vary more than ± 5% of the mean flow rate.
- No. of organisms per vessel/compartment: 5
- No. of vessels per concentration (replicates): 2 replicates consisting of four testing compartments (i.e. 20 daphnids in each treatment and control group)
- No. of vessels per control (replicates): 2 replicates consisting of four testing compartments (i.e. 20 daphnids in each treatment and control group)
- Test initiation: The juvenile daphnids were collected from the cultures and randomly transferred one or two at a time to transfer chambers until each chamber contained 5 daphnids. Each group of neonates then was randomly assigned to a control or treatment group and the neonates were transferred to the test compartments to initiate the test. All transfers were made below the water surface using wide-bopre pipettes.
- Other: Delivery of test solutions to the test chambers was initiated 6 d prior to introduction of the test organisms to the test water in order to achieve equilibrium of the test substance. The delivery system and the test chambers were placed in a temperature-controlled environmental chamber.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for culturing and testing was freshwater obtained from a well approximately 40 m deep located on the testing facility site. The well water was passed through a sand filter to remove particles greater than approximately 25 µm and pumped into a 37800 L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- Total organic carbon: < 1.0 mg/L (measured monthly, n = 1)
- Particulate matter: The water was filtered through 0.45 µm to remove fine particles prior use.
- Metals: < LOQ, except Calcium (36.2 mg/L), Magnesium (14.0 mg/L), Potassium (7.06 mg/L), and Sodium (18.1 mg/L)
- Pesticides: < LOQ
- Chloride: 4.3 mg/L
- Alkalinity: 176 mg/L as CaCO3
- Specific conductance: 356 µS/cm (n = 4)
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: The specific conductance, hardness, alkalinity, pH and total organic carbon (TOC) content of the well water was measured during a four-week period immediately preceding the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light and 8 h dark with a 30 min transition period of low light intensity
- Light intensity: 850 lux at the surfact of the water at test initiation provided by ambient laboratory light (fluorescent light bulbs with wavelengths similar to natural sunlight)

EFFECT PARAMETERS MEASURED:
1st generation daphnids:
- Immobilization: Daily
- Clinical signs of toxicity: Daily
- Presence of eggs/males/ephippia: Daily
- Body length & dry weight: At the end of the test
- Number of neonates produced by the 1st generation: With the onset of reproduction, neonates were counted and then discarded every Monday, Wednesday and Friday during the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approximately 2.5
- Range finding study: Yes, non-GLP
- Test concentrations: Control, 2.4, 8.1, 27, 90, and 300 µg a.i./L
- Results used to determine the conditions for the definitive study: Yes, 100% immobility at the highest two concentrations and thus no young produced.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

9.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

and survival, and growth (length & dry weight).

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

26 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

and survival

Details on results:

- Other biological observations: See section "Any other information on results incl. tables"
- Mortality of control: After 21 d of exposure, survival in the negative control group was 85%.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No, the test solutions in the mixing chambers and test chambers appeared clear and colorless throughout the test, with no evidence of precipitation.

Reported statistics and error estimates:

All statistical tests were performed with SAS software.
Survival data (discrete-variable data) were analyzed by Chi-square and Fischer's Exact test to identify treatment groups that showed a statistically significant difference (alpha = 0.05) from the control. All continuous-variable data were evaluated for normality using Shapiro-Wilk's test and for homogeneity of variance using Levene's test (alpha = 0.01). When the data passed the assumptions of normality and homogeneity, those treatments that were significantly different from the control means were identified using Dunnett's one-tailed test (alpha = 0.05).
The NOEC and LOEC for the production rate of the first brood were determined using Jonckheere-Terpstra step down trend test (alpha = 0.05).
EC50 values were determined for survival and reproduction endpoints and an EC10 value was estimated for the reproduction endpoint using a regression model. The EC50 value based on survival and the EC10 and EC50 values based on reproduction and growth endpoints were estimated using a Bruce-Versteeg regression model. The percent inhibition of each endpoint as compared to the control were also calculated and reported.

VALIDITY CRITERIA

The study fulfilled the criteria laid down by the guideline (Table 1) and is thus considered valid.

 

Table 1: Validity criteria for OECD 211.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals (female Daphnia) does not exceed 20% at the end of the test.	Mortality in the negative control was 15%.	Yes.
The mean number of living offspring produced per parent animal surviving at the end of the test is ≥ 60.	The mean number of living offspring produced per parent control animal surviving at the end of the test was 214.	Yes

Note: The same validity criterion (20%) can be used for accidental and inadvertent parental mortality for the controls as well as for each of the test concentrations.

 

ANALYTICAL RESULTS

Nominal concentrations selected for use in the study were 2.3, 5.8, 14, 36 and 90 µg a.i./L. The test solutions were clear and colorless throughout the test and there was no evidence of precipitation.

The measured test item concentrations in the sampled test solutions ranged from 12.6 to 109% of nominal concentrations. When the first generation daphnids in the 36 and 90 µg a.i./L treatment group reached 100% immobility on Days 18 and 9, respectively, each concentration had percent recoveries of 37.7 and 58.5%, respectively (Table 2).

The results of the study are based on the arithmetic mean values.

 

Table 2. Measured test item concentrations in test solution samples.

Nominal test item concentration	Sampling time	Measured concentration^1	Percent of nominal	Arithmetic Mean Measured Concentration^2	Arithmetic mean measured percent of nominal^2
[µg a.i./L]	[days]	[µg a.i./L]	[%]	[µg a.i./L]	[%]
Negative control	0	46.3% of LOQ	--	< LOQ	--
	6	< 30% LOQ	--
	14	< 30% LOQ	--
	14	< 30% LOQ	--
	21	< 30% LOQ	--
2.3	0	1.79	77.7	1.0	45
	6	1.44	62.7
	14	0.289	12.6
	14	0.348	15.1
	Mean	0.319
	21	0.575	25.0
5.8	0	5.21	89.8	3.4	58
	6	5.15	88.8
	14	0.940	16.2
	14	0.850*	14.7
	Mean	0.900		9.5	68
	21	2.19	37.7
14	0	15.2	108
	6	13.2	94.3
	14	5.11	36.5
	14	1.95	14.0
	Mean	3.53
	21	5.94	42.4
36	0	38.1	106	26	73
	6	39.2	109
	14	14.1	39.2
	14	15.4	42.9
	Mean	14.8
	18	13.6	37.7
90	0	93.4	104	78	87
	6	87.8	97.5
	9	53.0	58.5

¹The LOQ was 1.0 µg a.i./L

²Results were generated using Excel 2010.

* Since the response was below the response of the lowest standard concentration, the result was extrapolated.

 

 

BIOLOGICAL RESULTS

Survival and reproduction were the most sensitive biological endpoints measured in the study. After 21 d of exposure, there were no statistically significant treatment-related effects on survival and reproduction and growth at concentrations ≤ 9.5 µg a.i./L. There was no statistically significant difference in production rate of the first brood concentration at concentrations ≤ 26 µg a.i./L.

Daphnids exposed at concentrations ≥ 26 µg a.i./L had statistically significant reductions in survival and reproduction in comparison to the negative control. Consequently, the NOEC, based on survival and reproduction, was 9.5 µg a.i./L and the LOEC was 26 µg a.i./L. The effect values are summarized in Table 3.

 

Table 3. The 21-day EC50 value for adult survival / immobility and EC10 and EC50 based on reproduction and growth endpoints.

Parameter	EC10 (95% confidence interval) [µg a.i./L]	EC50 (95% confidence interval) [µg a.i./L]
Survival	NA	16 (NC)
Mean n° of neonates/reproductive day^1	15 (8.5 – 26)	23 (20 – 26)
Mean n° of live neonates at the end of the test per adult at start	12 (7.1 – 19)	19 (15 – 23)
Length	24 (1.5 - >78)	>78
Dry weight	8.2 (2.1 – 32)	35 (NC)

¹Reproductive days were defined as the number of days that the adult daphnid was alive from the day the first brood was released from any adult daphnid in the test until test termination. If an adult daphnid died, the number of reproductive days, for that adult, ended on the last day it was alive.

NA = not applicable; it was not required

NC = not calculated; the 95% confidence interval was overly wide and/or outside of the data used for calculation.

 

Detailed results

 

Survival. After 21 days of exposure, survival in the negative control group was 85%. Survival in the 1.0, 3.4, 9.5, 26 and 78 µg a.i./L treatment groups was 85, 100, 90, 0 and 0% at test end. The decrease in survival in the 26 and 78 µg a.i./L treatment groups were statistically significant (Fischer’s Exact) in comparison to the negative contro (p ≤ 0.05, Tables 4 and 5). Consequently, the NOEC for survival was 9.5 µg a.i./L and the LOEC was 26 µg a.i./L (Table 3). Based on the survival/ immobilities observed in the treatment groups, the 21-day EC50 value was 16 µg a.i./L, the 95% confidence interval was overly wide and not reported.

Daphnids in the negative control and the 1.0, 3.4 and 9.5μg a.i./L treatment groups that survived to test termination generally appeared normal. In the 26 and 78μg a.i./L treatment groups the first-generation daphnids were considered to be small in stature, lethargy and/or pale during the test prior to 100% immobility on Days 18 and 9, respectively.

 

Reproduction. Reproduction in the 78 µg a.i./L treatment group was not analyzed since no adults survived the test termination. The adult daphnids in this treatment group did not produce any neonate prior to 100% immobility on Day 9 of the test. The 1^st day of brood production in the negative control replicates and in all treatment replicates was on Day 8 or 9, indicating that there was no apparent delay in the onset of production with exception to one replicate in the 26 µg a.i./L treatment group (Day 11). No males or ephippia were produced during the test.

There was a statistically significant difference in the reproduction rate of the 1^stbrood at the concentration > 26 µg a.i./L treatment group compared to the control (Jonckheere-Terpstra step down trend test, p ≤ 0.05, Tables 4 and 5).

There was a statistically significant decrease in mean neonate production per reproductive day in the 26 µg a.i./L treatment groups compared to the negative control (Dunnett’s test, p ≤ 0.05, Tables 4 and 6). Reproductive days were defined as the number of days that the adult daphnid was alive from the day the 1^st brood was released from any adult daphnid in the test until test termination. Based on the mean number of live young produced per reproductive day observed in the treatment groups, the EC10 (21 d) was 15 µg a.i./L with a 95% confidence interval of 8.5 to 26 µg a.i./L and the EC50 (21 d) was 23 µg a.i./L with a 95% confidence interval of 20 to 26 µg a.i./L. Based on the mean number of live young produced at the end of the test per adult at the beginning of the test, the EC10 (21 d) was 12 µg a.i./L with a 95% confidence interval of 7.1 to 19 µg a.i./L and the EC50 (21 d) was 19 µg a.i./L with a 95% confidence interval of 15 to 23 µg a.i./L. Consequently, the NOEC for reproduction was 9.5 µg a.i./L and the LOEC was 26 µg a.i./L (Table 3).

 

Growth (mean length and mean dry weight). There were no statistically significant decreases in mean length or mean dry weight in any of the treatment groups compared to the negative control (Dunnett’s test, p > 0.05). There were no growth data for the 26 and 78 μg a.i./L treatment groups due to 100% immobility on Days 18 and 9, respectively (Tables 4 and 5). Consequently, the NOEC (21 d) for growth (length and dry weight) was 9.5 µg a.i./L and the LOEC (21 d) was > 9.5 µg a.i./L. The EC10 (21 d) and EC50 (21 d) for growth are provided in Table 3.

 

Table 4. Summary of Survival, Reproduction and Growth of Daphnia magna after 21 d exposure.

Arithmetic mean measured test item concentration	Percent adult survival	Mean production rate of first brood ± S.D.^1	Mean n° neonates per reproductive day ± S.D.	Mean n° neonates per adult at beginning of test ± S.D.	Mean length ± S.D.^2	Mean dry weight ± S.D.^2
[µg a.i./L]	[%]				[mm]	[mg]
Control	85	0.1255 ± 0.0091	16.4 ± 1.7	216 ± 20	4.9 ± 0.096	1.01 ± 0.061
1.0	85	0.1294 ± 0.0078	16.8 ± 0.85	200 ± 47	5.0 ± 0.050	1.12 ± 0.29
3.4	100	0.1294 ± 0.0078	15.1 ± 0.70	211 ± 9.1	5.0 ± 0.058	1.03 ± 0.087
9.5	90	0.1294 ± 0.0078	15.7 ± 0.55	202 ± 21	4.9 ± 0.10	0.93 ± 0.123
26	0*	0.1238 ± 0.019	5.83 ± 1.5*	35.5 ± 9.5*	--^4	--^4
78	0*	--^3	0.0^3	--^3	--^4	--^4
EC10 (95% CI)	NA	NA	15 (8.5 - 26)	12 (7.1 - 19)	24 (1.5 - >78)	8.2 (2.1 - 32)
EC50 (95% CI)	16 (NC)	NA	23 (20 – 26)	19 (15 – 23)	>78 (NC)	35 (NC)

¹There were no significant differences from the negative control (Jonckheere-Terpstra step down trend test, p > 0.05)

²No statistically significant difference in growth found in any of the treatment groups from the negative control (Dunnett’s one-tailed test, p > 0.05)

³No data because the first generation daphnids in the treatment group did not reach reproductive stage.

⁴No growth data due to 100% immobility prior to test termination.

*Statistically significant reduction in percent survival of the first generation daphnids at test termination (Fischer’s Exact test, p ≤ 0.05) and in mean number of live neonates produced per reproductive day and mean number of live neonates produced per adult at the beginning of the test (Dunnett’s one-tailed test, p ≤ 0.05) from the negative control.

NA = Not applicable, not required; NC = Not calculated, the 95% confidence interval values were overly wide and/or outside of the data used for the calculation.

 

Table 5. Percent Inhibition compared to the negative control

Arithmetic mean measured concentration	Percent adult survival	Percent inhibition	Mean production rate of 1^stbrood ± S.D.	Percent inhibition	Mean n° neonates per repro. Day^3± S.D.	Percent inhibition	Mean n° neonates per adult at test start ± S.D.	Percent inhibition	Mean length ± S.D.	Percent inhibition	Mean dry weight ± S.D.	Percent inhibition
[µg a.i./L]	[%]	[%]		[%]		[%]		[%]	[mm]	[%]		[%]
control	85	--^1	0.1255	--^1	16.4	--^1	216	--^1	4.9	--^1	1.01	--^1
1.0	85	0	0.1294	-3.11	16.8	-2.44	200	7.41	5.0	-2.04	1.12	-10.89
3.4	100	-17.6	0.1294	-3.11	15.1	7.93	211	2.31	5.0	-2.04	1.03	-1.98
9.5	90	-5.9	0.1294	-3.11	15.7	4.27	202	6.48	4.9	0.0	0.93	7.92
26	0	100	0.1238	1.35	5.83	64.45	35.5	83.56	--^2	--^2	--^2	--^2
78	0	100	--^2	--^2	--^2	--^2	--^2	--^2	--^2	--^2	--^2	--^2

¹Not applicable

²No data available due to 100% immobility.

³Reprodcutive days were defined as the number of days that the adult daphnid was alive from the day the first brood was released from any adult daphnid in the test until test termination. If an adult daphnid died, the number of reproductive days, for that adult, ended on the last day it was alive.

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

NOEC (21 d) = 9.5 µg a.i./L (arithmetic mean measured, OECD 211, D. magna, reproduction/survival/growth)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

9.5 µg/L

Additional information

There is one GLP study available, in which the long-term toxicity ofsodium dimethyldithiocarbamate (CAS128-04-1)to aquatic invertebrates was assessed according to OECD guideline 211.

 

In a continuous flow-through exposure system with a water renewal rate of approximately 8 volume additions of test water in each test chamber per day, Daphnia magna was exposed to five nominal test item concentrations in a geometric series consisting of 2.3, 5.8, 14, 36, and 90 µg a.i./L test item for 21 d. The effects of the test item were assessed in terms of survival, reproduction and growth (mean length and mean dry weight) to determine the NOEC and LOEC values. EC10 values were calculated for reproduction and growth and EC50 values were calculated for reproduction, growth and for 1^stgeneration immobility at test termination, when possible. The actual test item concentrations were analytically verified by GC/MS at the beginning of the test, at approximately weekly intervals during the test and at the end of the test.

The measured test item concentrations during the test ranged from 12.6 to 109% of nominal. When the first generation daphnids in the 36 and 90 µg a.i./L treatment groups reached 100% immobility on Days 18 and 9, respectively, each concentration had recoveries of 37.7 and 58.5%, respectively. Therefore, results were based on the calculated arithmetic mean measured concentrations of 1.0, 3.4, 9.5, 26, and 78 µg a.i./L.

Survival and reproduction were the most sensitive biological endpoints measured in this study. After 21 d, there were no statistically significant treatment-related effects on survival, reproduction and growth at concentrations ≤ 9.5 µg a.i./L and there was no statistically significant difference in the production rate of the 1^stbrood at concentrations ≤ 26 µg a.i./L. However, daphnids exposed to test item concentrations of ≥ 26 µg a.i./L had statistically significant reductions in survival and reproduction compared to the negative control. Thus, the derived NOEC (21 d) based on survival and reproduction, was 9.5 µg a.i./L and the LOEC (21 d) was 26 µg a.i./L.