4,4'-methylene bis(dibutyldithiocarbamate)

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Remarks:

(dietary exposure)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 August 2019 - 26 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -III: Dietary Exposure Bioaccumulation Fish Test

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Radiolabeled test material: 4,4’-methylene bis(dibutyldithiocarbamate), [CH2-14C]
Non-Radiolabeled test material (for co-chromatography): 4,4’-methylene bis(dibutyldithiocarbamate)
CAS Number: 10254-57-6
Physical state: Amber-green colored liquid

Radiolabelling:

yes

Details on sampling:

SAMPLING INTERVALS/FREQUENCY FOR TEST ORGANISMS

On Day 0 (uptake), five individual fish were randomly sampled from the main stock tank for analysis. On Day 14 (uptake), five individual fish were randomly sampled from the control and ten individual fish were sampled from the test group. On Days 1, 4, 7, 14 and 21 of the depuration phase, five fish were taken from the control and treatment tanks for analysis, on Day 28 (depuration) ten fish were taken from the control and treatment tanks for analysis. Fish were blotted dry and measured for length and width. Samples were shipped on the day of sampling under chilled conditions, except for the time zero fish which were received frozen.

EXTRACTION OF FISH FOR ANALYSIS

At least duplicate aliquots were taken for analysis and concentrations of radioactivity were determined by combustion and radioassay. Samples for total radioactivity measurements were generally analyzed on the day of sampling, where possible, or stored at approximately 2 - 8 deg C.

Fish were pooled (by group and sampling occasion), homogenised by mincing with scissors, and extracted with four portions of acetonitrile (10 mL) by mixing with a Silverson blender for 2 minutes, followed by sonication for 5 minutes. The extracts were separated by centrifugation (2500 rpm for 15 minutes) and the total weight recorded. Duplicate weighed aliquots (50 µL) were taken for radioassay. The post extraction solids were dried, weighed and triplicate weighed aliquots (approximately 0.1 g) taken for combustion and radioassay. For each sample, portions of the extract solutions were combined in proportion to their total weight. The pooled extracts were analyzed by reverse phase high pressure liquid chromatography (HPLC).

The lipid content was measured in five fish taken from the control tank and pooled on each sampling occasion of the exposure and depuration phases. Sub samples (ca 5 g) of the homogenised pooled fish were extracted with chloroform and methanol (1:2; 5 mL) followed by chloroform (5 mL) and water (5 mL). After each solvent addition the samples were homogenised with an Ultra-turax blender. Samples were centrifuged at 3000 rpm for 15 minutes and the chloroform layer transferred to a pre-weighed glass vial. The chloroform was evaporated to dryness in an oven at ca. 55 deg C and the weight of the dried residue (lipid) was determined.

EXTRACTION OF CONTROL AND TREATED FEED FOR ANALYSIS

Samples of the treated feed were taken for analysis on Days 0 and 14 (uptake). From the control and treated feed subsamples five weighed aliquots (approximately 0.025 to 0.1 g) were taken for combustion and radioassay, respectively. From the treated feed subsamples, a portion (3 g) was extracted with four portions (10 mL) of acetonitrile by vortex mixing for 1 minute, followed by sonication for 15 minutes. The extract was separated by centrifugation (3000 rpm for 15 minutes) and the volume recorded. Duplicate aliquots (50 µL) were removed for radioassay. The post extraction solid was dried, weighed and triplicate weighed aliquots (approximately 0.1 to 0.15 g) were taken for combustion and radioassay. For each sample extracted, portions of the extract solution were combined in proportion to their total volume. The pooled extracts were analyzed by reverse phase HPLC.

HOMOGENEITY AND STABILITY OF TREATED FEED

Commercial diet treated at nominal concentrations of 10, 100 and 1000 µg/g were analyzed to confirm homogenous distribution of radioactivity on the feed. Five replicate aliquots (approximately 0.1 g) were analyzed by combustion and radioassay.

The feed treated at a nominal concentration of 100 µg/g was analyzed to assess stability. A subsample of the treated feed (approximately 5 g) was extracted with portions of acetonitrile (2 or 4 × 10 mL) immediately following application and after 4, 7, 10, 17 and 20 days of frozen storage. The extract was vortex mixed for 1 minute, followed by sonication for 15 minutes. The extract was separated by centrifugation (3000 rpm for 15 minutes) and the volume measured. Duplicate aliquots (50 µL) were removed for radioassay. The post-extraction solid was dried, weighed and triplicate weighed aliquots (approximately 0.1 g) taken for combustion and radioassay.

Portions of the extract solution were combined in proportion to their total volume. The pooled extracts were analyzed by reverse phase high performance liquid chromatography (HPLC).

LIPID ANALYSIS OF TREATED FEED

The lipid content of the treated feed was not confirmed analytically in this study. The kinetic analysis therefore was performed using a range of default food lipid content values, 15% - 20%, to bracket the estimated average lipid content of the treated feed prepared with corn oil.

Vehicle:

yes

Remarks:

dietary exposure (feed prepared with corn oil).

Details on preparation of test solutions, spiked fish food or sediment:

The fish were exposed to 4,4’-methylene bis(dibuytldithiocarbamate) in a commercial diet treated at a nominal concentration of 1000 µg/g. The test material was radio-diluted with non-radiolabeled test material prior to administration of the test material to the diet. A portion of radiolabeled test material (19.53 mg, 87.56 MBq) was transferred to a volumetric flask (5 mL) and evaporated to dryness under nitrogen. A portion of a non-radiolabeled test material (155.57 mg, 3.81 mL), diluted to volume with ethanol, manually shaken and sonicated for one minute. A portion (4.28 mL) of the radiodiluted stock solution was added to corn oil (3.0 mL) and the ethanol removed under nitrogen. The treated corn oil was added to the fish feed, and mixture was manually shaken followed by rotational agitation for five minutes. This was repeated five times. The feed was separated into three portions and stored frozen.

Control diet was prepared using a portion of feed treated only with corn oil, and the mixture was manually shaken followed by rotational agitation for five minutes. This was repeated five times. The control feed was stored frozen untill used.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Rainbow Trout were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK. The fish were acclimatized in holding tanks for a period of at least 2 weeks and fed commercial trout pellets . No mortality was observed during the acclimatization period. Prior to initiation of exposure in the main test, the weight and lenght was determined for a subsample of 10 fish; selected at random rom the batch to be used in the test.

Route of exposure:

feed

Justification for method:

dietary exposure method used for following reason: Low aqueous solubility of the test material.

Remarks:

Dose level selected was the highest concentration tested at which no adverse effects were observed in fish, all food was consumed and accumulated levels of radioactivity were sufficient to monitor 95% reduction in body burden.

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

14 d

Total depuration duration:

28 d

Hardness:

Control: 184 mg CaCO3 (Day -1); 168 mg CaCO3 (Day 0)
1000 ug/g: 188 mg CaCO3 (Day -1); 164 mg CaCO3 (Day 0)

Test temperature:

Control: 13.5 - 14.3°C
1000 ug/g: 13.8 - 14.4°C

pH:

Control: 7.6 - 8.3
1000 ug/g: 7.6 - 8.2

Dissolved oxygen:

Control: 8.9 - 10.2 mg/L (86 - 99% ASV)
1000 ug/g: 7.3 - 10.3 mg/L (71 - 100% ASV)

TOC:

<1 mg C/L (LOQ), measured on Day -1

Details on test conditions:

Two glass aquaria (one test and one control), fitted with overflows to maintain a volume of approximately 100 liters, were set up with a continuous flow through of approximately 1152 liters per day (nominal flow rate 800 mL/minute). Laboratory tap water (also used for the preliminary palatability study) was dechlorinated by passage through an activated carbon filter (Purite Series 500). A proportion of the incoming water was softened (Elga Nimbus 1248D Duplex water softener) and then remixed with the main supply to give a water hardness of approximately 140 mg/L as CaCO3, and a pH of 6.0 to 8.5. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

The temperatures in the test tanks were maintained at approximately 14˚C. The water was aerated to maintain oxygen levels equal to or greater than 60% oxygen saturation. A lighting regime of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods was maintained.

The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 13°C to 17°C with a maximum deviation of ±2°C and a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 14 days for the uptake phase.

Nominal and measured concentrations:

Nominal concentration: 1000 µg/g;
Measured concentrations: 919 µg/g (Day 0) and 920 µg/g (Day 14, uptake)

Reference substance (positive control):

not required

Details on estimation of bioconcentration:

Non-linear regression of mean concentrations (as radioactivity) in whole rainbow trout following 14 days of dietary administration of radiolabeled test material at a nominal concentration of 1000 µg/g, followed by 28 days of depuration were used in the kinetic evaluation to determine bioaccumulation parameters. The kinetic dietary biomagnification factor (BMFk) was determined to be 0.129. The growth corrected half-life (t1/2) was determined to be 24.1 days. The fish lipid normalized biomagnification factor corrected for growth rate (BMFkgL) was determined to be between 0.777 (based on 15% lipid food content) and 1.04 (based on 20% lipid food content).

Lipid content:

> 4.89 - < 7.42 %

Time point:

end of exposure

Remarks on result:

other: in fish

Lipid content:

> 15 - < 20 %

Remarks on result:

not measured/tested

Remarks:

(in feed; estimated based on average lipid content in feed and corn oil)

Conc. / dose:

1 000 µg/g food

Temp.:

14 °C

pH:

8

Type:

BMF

Value:

0.129 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic

Key result

Conc. / dose:

1 000 µg/g food

Temp.:

14 °C

pH:

8

Type:

BMF

Value:

0.182 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic, corrected for growth

Key result

Conc. / dose:

1 000 µg/g food

Temp.:

14 °C

pH:

8

Type:

BMF

Value:

0.777 dimensionless

Basis:

normalised lipid fraction

Remarks:

(fish lipid normalized; corrected for 15% lipid food content)

Calculation basis:

kinetic, corrected for growth

Key result

Conc. / dose:

1 000 µg/g food

Temp.:

14 °C

pH:

8

Type:

BMF

Value:

1.04 dimensionless

Basis:

normalised lipid fraction

Remarks:

(fish lipid normalized; corrected for 20% lipid food content)

Calculation basis:

kinetic, corrected for growth

Key result

Rate constant:

growth-corrected half-life (d)

Value:

24.1

Remarks on result:

other: estimate

Details on kinetic parameters:

Mean concentrations of radioactivity in whole rainbow trout (expressed in terms of µg equivalents of radioactivity per g) following 14 days of dietary administration of the test material at a nominal concentration of 1000 µg/g and 28 days depuration.

Metabolites:

Chromatographic analysis of fish extracts during both exposure and after 4 days of depuration indicated the presence of four low level, unidentified metabolites each accounted for <=3.4% of the sample radioactivity. The low level metabolites were not detected in extracts after 7 days depuration. As none of these metabolites accounted for <10% of the sample radioactivity, no further analysis was conducted on these components.

Details on results:

No mortalities or sub lethal effects of exposure were observed following exposure of fish to commercial diet treated with radiolabeled test material at a nominal concentration of 1000 µg/g, over a 14-day exposure period followed by a depuration period of 28 days, under flow-through conditions at 14 deg C.

The achieved concentration of radiolabeled test material was confirmed by direct analysis of the bulk treated feed to be 869 µg/g with the homogeneity within ±15% of the mean. The extractability of radioactivity from the treated feed was 99.6 and 101.8% of applied radioactivity. Chromatographic analysis of the feed extracts showed that test material accounted for 98.3 and 98.6% sample radioactivity, confirming the stability of the test item on feed for the duration of the 14 day exposure phase.

After 14 days of exposure to radiolabeled test material, the mean concentration of total radioactivity in fish tissue was 71.4 µg equivalents/g. During the depuration phase, approximately 40% of the accumulated radioactivity was eliminated by Day 4 of depuration, and by Day 28 of depuration, approximately 70% radioactivity had been eliminated.

Chromatographic analysis of fish extracts indicated that parent test material accounted for 87.0% of the sample radioactivity (71.4 µg/g) after 14 days of exposure. Following 28 days of depuration, parent test material accounted for 96.6% of the sample radioactivity (19.9 µg/g), suggesting that any metabolites formed may have been preferentially eliminated.

Chromatographic analysis of fish extracts during both exposure and after 4 days of depuration indicated the presence of four low level, unidentified metabolites that each accounted for ≤3.4% of the sample radioactivity. The low level metabolites were not detected in extracts after 7 days depuration. As none of these metabolites accounted for <10% of the sample radioactivity, no further analysis was conducted on these components.

Kinetic analysis estimated the biomagnification factor (BMFk) to be 0.129 and the lipid normalized biomagnification factor corrected for growth rate (BMFkgL) to be between 0.777 (based on 15% lipid food content) and 1.04 (based on 20% lipid food content). The growth corrected half-life (t1/2g) was estimated to be 24.1 days.

Reported statistics:

Radioactivity concentration-time data in whole fish were analyzed using the software R version 3.6.3 (2020-02-29) (The R Foundation for Statistical Computing) and OECD-TG305 R-Package bcmfR (v0.4-18) consistent with OECD 305 Guidance (2017).

Model fitting and simulations were performed using estimated lipid food contents of 15% and 20%, as analysis of the feed lot used on study was not performed.

Optimum model results were obtained using a log-transformed non-linear regression. Model diagnosis (see Figure 1) suggests that the model residuals are normally distributed with a Shapiro-Wilk normality test p-value of 0.8603.

Validity criteria fulfilled:

yes

Conclusions:

Fish were exposed to radiolabeled test material at a nominal concentration of 1000 ug/g in feed over a 14 day exposure period. At the end of the 14 day exposure period, 10 fish were removed for analysis. The depuration of accumulated residues in the remaining fish was monitored over 28 days. The mean concentration of total radioactivity in fish tissue was 71.4 ug equivalents/g after 14 days of exposure, but declined to 19.9 ug equivalents/g. A reduction in total body burden of 72.1% was oberved after 28 days of depuration, which constituted an elimination of radioactivity of approximately two half lives of that which was accumulated.

The amount of test material extracted from the fish was in the range 92.0 – 104.2% sample radioactivity. Chromatographic analysis of the extracts showed the majority of radioactivity extracted from fish tissue was unchanged test material, and after 14 days of exposure accountinged for 87.0% sample radioactivity (71.4 µg/g) after 14 days of exposure. After 28 days depuration, the test material accounted for 96.6% sample radioacitivity (19.9 µg/g).

Four low level, unidentified metabolites were observed after 14 days exposure. These metabolites were present at Day 1 and 4 of depuration and not detected in extracts after 7 days of depuration.

For the duration of the depuration phase, the overall range of lipid content measured was 4.89 - 7.42%. The mean concentrations of the test material normalized to an average of 5% lipid content declined rom 54.9 ug equivalents/g after 14 days of exposure to 14.7 ug equivalents/g after 21 days of depuration. The concentration remained at a similar level after 28 days of depuration.

The kinetic biomagnification factor (BMFk) and the fish lipid normalized biomagnification factor corrected for growth rate (BMFkg) were estimated to be <1. The fish lipid normalized biomagnification factor corrected for growth rate (BMFkgL) was estimated to be between 0.777 ( based on 15% lipid food content) and 1.04 (based on 20% lipid food content). The growth corrected half-life was estimated to be 24.1 days, suggesting a relatively slow elimination of radioactivity from fish tissues following a 14 day exposure to food treated with the test material.

Executive summary:

Rainbow trout (Oncorhynchus mykiss) were exposed to commercial diet treated with radiolabeled test material at a nominal concentration of 1000 µg/g over a 14-day exposure period followed by a depuration period of 28 days, under continous flow-through conditions at 14 deg C. No mortalities or sub lethal effects of exposure were observed through the test.

 

Radioactivity was accumulated in fish tissues following exposure to feed treated with the test material for 14 days. The kinetic biomagnification factor (BMFk) and the fish lipid normalized biomagnification factor corrected for growth rate (BMFkg) were estimated to be <1. The fish lipid normalized biomagnification factor corrected for growth rate (BMFkgL) was estimated to be between 0.777 ( based on 15% lipid food content) and 1.04 (based on 20% lipid food content). The growth corrected half-life was estimated to be 24.1 days, suggesting a relatively slow elimination of radioactivity from fish tissues following a 14 day exposure to food treated with the test material.

 

At Day 28 of the depuration phase, eliminated radioactivity accounted for approximately two half lives of that which had been accumulated.

 

The majority of radioactivity extracted from fish tissue was unchanged, parent material in the range of 87.0 - 97.2% sample radioactivity. Up to four low unidentified metabolites were detected, but these low level metabolites were not detected in extracts after 7 days of depuration.

Description of key information

Rainbow trout (Oncorhynchus mykiss) were exposed to commercial diet treated with radiolabeled test material at a nominal concentration of 1000 µg/g over a 14-day exposure period followed by a depuration period of 28 days, under continous flow-through conditions at 14 deg C. No mortalities or sub lethal effects of exposure were observed through the test.

 

Radioactivity was accumulated in fish tissues following exposure to feed treated with the test material for 14 days. The kinetic dietary biomagnification factor (BMFk) was determined to be 0.129. The fish lipid normalized biomagnification factor corrected for growth rate (BMFkgL) was determined to be between 0.777 (based on 15% lipid food content) and 1.04 (based on 20% lipid food content). The growth corrected half-life (t1/2) was determined to be 24.1 days.

 

At Day 28 of the depuration phase, eliminated radioactivity accounted for approximately two half lives of that which had been accumulated.

 

The majority of radioactivity extracted from fish tissue was unchanged, parent material in the range of 87.0 - 97.2% sample radioactivity. Up to four low unidentified metabolites were detected, but these low level metabolites were not detected in extracts after 7 days of depuration.

Key value for chemical safety assessment

BMF in fish (dimensionless):

0.777

Additional information

Kinetic dietary biomagnification factor (BMF_k):  0.129

Fish lipid normalized biomagnification factor corrected for growth rate (BMF_kgL):  0.777 (15% lipid food content) - 1.04 (20% lipid food content)

Growth corrected half-life (t_1/2):  24.1 days