Nickel bis(dibutyldithiocarbamate)

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Mar 2018 to 27 Apr 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Nickel Dibutyldithiocarbamate (NDBC)
- Physical Description: Solid
- Storage Conditions: Ambient
- Batch No: 70900202
- Measured Carbon Content: 45.75% (As determined by elemental analysis)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: The Easton Wastewater Treatment Facility, Easton, Maryland on March 26, 2018.
- Preparation of inoculum for exposure: The sludge was sieved using a 2-mm screen and adjusted to approximately 1000 mg total suspended solids/L with mineral media and then aerated at test temperature until use.
- Pretreatment: A total suspended solids measurement and standard plate count were performed on the inoculum on the day of use in the test. Plates were incubated at 20 ± 3 ºC for approximately 48 hours.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Test temperature: 19.15 - 21.60 °C; room temperature was monitored continuously using an automatic data logger.
- pH: 7.52 - 7.60
- pH adjusted: No

TEST SYSTEM
- Culturing apparatus: The test chambers were amber 4-liter bottles.
- Aeration: The air entering the chambers was passed through Drierite™ to remove ambient moisture and then through Ascarite® to produce CO2-free air.
- Details of trap for CO2 and volatile organics if used: The air exiting the test chambers was passed through a series of three gas washing bottles, each containing approximately 100 mL of 0.5 M potassium hydroxide (KOH) to trap the CO2 that had evolved within the chamber. An additional set of gas washing bottles that was not connected to a chamber was maintained concurrently with the traps connected to the chambers. These bottles contained approximately 100 mL of 0.5 M KOH and the amount of CO2 detected in the KOH solution was subtracted from the CO2 in the control traps to determine the amount of CO2 produced by the inoculum in the blank control.
- Other: Magnetic stir bars and stir plates were used to mix the contents of the test chambers. Stir plate motors were cycled on and off approximately every 15 minutes to prevent the heating of the stirrer motors.

COMPOSITION OF TEST MEDIUM
The following were added to each chamber:
- 2470 mL of high grade water
- 3 mL calcium chloride solution (2.75%)
- 3 mL of ferric chloride solution (0.025%)
- 3 mL of magnesium sulfate solution (2.25%)
- 30 mL of phosphate buffer (pH 7.4)
- A volume of activated sludge soil-amended inoculum to achieve a final TSS concentration of ≤30 mg/L
Reference substance stock solution (74.5 mL) was added to the appropriate chambers to achieve a nominal concentration of 10 mg C/L. Test substance was weighed out (0.0656 g) and added to the appropriate chambers to achieve a nominal concentration of 10 mg C/L. The final volume within all chambers was brought up to 3000 mL by the addition of GenPureTM water.

PREPARATIONS
The following test assays were prepared:
- 3 blank control assays
- 3 test substance assays
- 3 inhibition control assay
- 1 toxicity control

TEST INITIATION
The biodegradation test was started by bubbling CO2-free air through each of the test chambers at a rate of approximately 55 mL per minute. The CO2 produced from the degradation of organic carbon sources within the test chamber was trapped as potassium carbonate (K2CO3) in the KOH solution and the amount of inorganic carbon in the trapping solution was measured at various intervals during the study, using a Shimadzu Model TOC-VCSH carbon analyzer. The test substance was administered to the treatment group and toxicity control test chambers by direct weight addition.

TEST TERMINATION
On the 28th day of the test, an aliquot of the contents of each test chamber was removed and the pH determined. The contents of all chambers were then acidified by the addition of 1 mL of concentrated hydrochloric acid to drive off inorganic carbonate. All chambers were aerated overnight and then a sample from each test chamber was removed for dissolved organic carbon (DOC) analysis and the
trapping solutions closest to the test chambers were analyzed for inorganic carbon.

Reference substance:

benzoic acid, sodium salt

Remarks:

10 mg C/L

Test performance:

VALIDITY CRITERIA
- The amount of CO2 evolved by the blank control chambers did not exceed the 40 mg/L (120 mg total).
- Deviation of the degree of biodegradation of the test substance in the plateau phase was <20%.
- The degree of biodegradation of the reference substance was >60% CO2/ThCO2 after 14 days.
- The degree of biodegradation in the inhibition control was >25 % CO2/ThCO2 after 14 days.
- The amount of produced CO2 in the inoculum blank (“blank controls”) at the end of exposure was <70 mg/L (mean value).

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Remarks on result:

other: test substance dosed test chambers evolved less CO2 compared with the blank control by Day 28 of the test

Details on results:

A detailed overview of the test results is provided in 'Any other information on restults incl. tables'.
- Blank controls: The blank control chambers evolved an average of approximately 84 mg CO2 over the test period, corresponding to 28 mg CO2/L. This value has been corrected for the amount of CO2 in the trapping solution since potassium hydroxide solution, even when freshly prepared, contains carbonates. The amount of CO2 evolved by the blank control chambers did not exceed the 40 mg/L (120 mg total) value considered the acceptable limit for CO2 evolution tests
- Test substance degradation: The cumulative CO2 produced in test chambers of the test substance was approximately 3 mg less than that of the non-dosed blank control, suggesting that the test substance was not degraded in activated sludge medium.
- Toxicity control: The toxicity control achieved > 25% degradation by Day 14 and therefore NDBC may be considered non-inhibitory at the concentration tested in this study.

Results with reference substance:

An average of 99.1% of theoretical CO2 was evolved over the test period. An average percent biodegradation of greater than 60% was achieved by Day 5.

Table: Measured Inorganic Carbon Concentration of Trapping Solutions (mg C/L)* 

Day	Blank Control 1	Blank Control 2	Blank Control 3	Sodium Benzoate Rep. 1	Sodium Benzoate Rep. 2	Sodium Benzoate Rep. 3	Test substance Rep. 1	Test substance Rep. 2	Test substance Rep. 3	Toxicity control	KOH blank
3	49.6	50.1	47.7	175.4	192.4	191.6	46.4	38.6	41.9	173.1	7.8
5	37.7	38.4	36.7	92.6	101.9	92.2	40.0	37.0	31.2	93.9	7.9**
8	49.0	48.0	50.3	91.5	88.1	89.4	48.0	46.4	45.8	95.7	7.3
12	45.4	47.2	47.6	76.1	66.8	73.4	46.4	48.9	48.5	77.8	7.6
15	26.4	25.5	24.7	36.2	32.6	38.2	25.1	27.0	25.8	35.8	7.1
18	22.8	22.3	21.6	28.4	25.9	30.2	23.0	23.6	22.1	31.1	7.1
22	20.4	21.7	23.3	26.6	25.5	44.7	21.8	23.4	22.0	26.1	8.4
26	20.1	21.2	31.2	25.5	26.3	29.1	22.5	22.1	20.5	24.4	8.3
29	20.0	22.3	25.5	28.2	28.0	31.5	25.3	24.7	24.1	13.6	7.7

* Values were rounded

* Value was extrapolated

 

Table: Cumulative Milligrams of Carbon Dioxide Evolved (1)(2)

Day	Blank Control 1	Blank Control 2	Blank Control 3	Sodium Benzoate(3) Rep. 1	Sodium Benzoate(3) Rep. 2	Sodium Benzoate(3) Rep. 3	Test substance(3) Rep. 1	Test substance(3) Rep. 2	Test substance(3) Rep. 3	Toxicity control	KOH blank
3	18.2	18.4	17.5	46.3	52.6	52.3	-1.0	-3.9	-2.6	45.5	2.9
5	32.0	32.5	31.0	66.5	76.2	72.3	-0.1	-4.1	-5.0	66.2	5.7
8	50.0	50.1	49.5	82.1	90.5	87.1	-0.5	-5.1	-6.2	83.3	8.4
12	66.7	67.4	66.9	92.9	97.8	96.9	-0.7	-4.3	-5.5	94.6	11.2
15	76.3	76.8	76.0	96.8	100.4	101.6	-0.9	-3.8	-5.4	98.4	13.8
18	84.7	84.9	83.9	99.1	101.8	104.5	-0.6	-3.3	-5.5	101.7	16.4
22	92.2	92.9	92.5	100.9	103.2	112.9	-0.6	-2.7	-5.4	103.2	19.5
26	99.6	100.7	103.9	101.3	103.9	114.7	-1.2	-3.5	-6.8	103.3	22.6
29	106.9	108.9	113.2	103.4	105.9	118.0	-0.2	-2.7	-6.2	100.0	25.4

1) The results of the inorganic carbon analyses of the CO2 traps were converted to mg CO2 produced using the following equation:

mg CO2 = cumulative result (mg C/L) × vol. of KOH (L) × 3.67 mg CO2/mg C

2) Calculations performed in Excel 2010 full precision mode. Manual calculations may differ.

3) Corrected for the CO2 attributed to the inoculum and the KOH by subtracting the average amount of CO2 evolved by the controls.

 

Table: Cumulative Percent of Theoretical Carbon Dioxide Evolved (1)

Day	Blank Control 1	Blank Control 2	Blank Control 3	Sodium Benzoate Rep. 1	Sodium Benzoate Rep. 2	Sodium Benzoate Rep. 3	Test substance Rep. 1	Test substance Rep. 2	Test substance Rep. 3	Toxicity control
3	NA	NA	NA	42.1	47.8	47.5	-0.9	-3.5	-2.4	20.7
5	NA	NA	NA	60.4	69.2	65.7	-0.1	-3.7	-4.5	30.0
8	NA	NA	NA	74.6	82.2	79.1	-0.5	-4.6	-5.6	37.8
12	NA	NA	NA	84.4	88.9	88.0	-0.6	-3.9	-5.0	43.0
15	NA	NA	NA	87.9	91.2	92.2	-0.8	-3.4	-4.9	44.7
18	NA	NA	NA	90.0	92.4	94.9	-0.5	-3.0	-5.0	46.2
22	NA	NA	NA	91.6	93.7	102.5	-0.5	-2.4	-4.9	46.9
26	NA	NA	NA	92.0	94.4	104.2	-1.1	-3.1	-6.1	46.9
29	NA	NA	NA	93.9	96.2	107.2	-0.2	-2.4	-5.6	45.4
Cumulative Average Day 29 (n=3)				99.1			-2.7			45.4
Standard Deviation				7.1			2.7

1) Calculations performed in Excel 2010 full precision mode. Manual calculations may differ.

NA – Not Applicable

Validity criteria fulfilled:

yes

Remarks:

see 'Test performance'

Interpretation of results:

not readily biodegradable

Description of key information

Not readily biodegradable according to OECD guidelines.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The biodegradation potential of the substance in water was determined in a screening study according to OECD TG 301B (CO2-Evolution Test) and in compliance with GLP criteria. In this study, 10 mg C/L nominal was inoculated with activated sludge from a municipal wastewater treatment plant for 28 days under aerobic conditions. During the incubation period the produced carbon dioxide (CO2) was measured and at the end of the test compared with the maximal theoretical CO2 production (ThCO2). Biodegradation was expressed as percentage of the measured CO2 relative to the ThCO2. After the 28-day incubation period <10% of the substance was biodegraded. Based on these findings, the test substance is classified as not readily biodegradable.