Zinc bis(dibenzyldithiocarbamate)

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Transformation products:

not measured

Reference 2

Endpoint:

biodegradation in water: sediment simulation testing

Data waiving:

other justification

Justification for data waiving:

other:

Transformation products:

not specified

Reference 3

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

18 May 2021 to 12 Apri 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Version / remarks:

April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

An overview of the characterisation of the test water is provided in Table 1 in 'Any other information on materials and methods incl. tables'.
- Details on collection: Collected from Tuckahoe Lake, MD (GPS: 38.968573 N, 75.944673 W) on 27 April 2021.
- Transportation: The water was transported in a sealed container with enough headspace to provide access to air and arrived at the test facility on 29 April 2021.
- Pre-treatment: Upon arrival, the water was filtered through a 0.2 mm sieve to remove coarse particulate matter and debris. Samples were prepared by measuring 100 mL of water via a graduated cylinder and dispensing into 250 mL amber bottles. Additionally, a portion of water was placed in the autoclave for 30 minutes at 250°F (15 psi) to be sterilized for the sterile sample set. The sterilized water was dispensed using sterilized graduated cylinders into 250 mL sterilized amber glass bottles (which had also been autoclaved at 250°F and 15 psi for 30 minutes).

Duration of test (contact time):

62 d

Initial conc.:

3.92 µg/L

Based on:

test mat.

Initial conc.:

9.86 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 100 mL
- Test temperature: 12 ± 2 °C
- Average pH: 7.28 (test substance); 7.37 (reference substance)
- Dissolved oxygen: 8.11 – 8.99 ppm
- Continuous darkness: Yes
- Others: Following application, all samples were placed on orbital shakers, connected to their respective traps for volatiles.

TEST SYSTEM
- Culturing apparatus: 250 mL amber bottles with teflon septa
- Number of culture flasks/concentration: 16
- Number of culture flasks/positive control: 6
- Number of culture sterile flasks dosed at the high dose rate: 8
- Details of trap for CO2 and volatile organics if used: Starting with the 14 day sampling, high dose, low dose, and reference control samples were evaluated for 14CO2 dissolved in the water layer. An aliquot (25 mL) of the sample water layer was taken prior to decanting/centrifugation and transferred to a 40 mL glass vial. This vial was connected to a water humidifier, a 10% sodium hydroxide trap (20 mL), and a peristaltic pump to pull air through the system. The sample water aliquot was acidified by addition of 6 N HCl (1 mL) through the septum cap and the sample allowed to evolve 14CO2 for 2 hours while bubbling air through the water layer. After evolving, volumes of the sample and trap were measured and triplicate aliquots taken (1 mL for aqueous sample, 0.5 mL for NaOH trap) for radioassay by LSC.

SAMPLING
High and low dose samples were sacrificed after 0, 7, 14, 28, 43, and 62 days of incubation. Sterile samples were sacrificed after 14, 43, and 62 days of incubation. Benzoic acid reference samples were sacrificed after 7 and 14 days of incubation. Duplicate samples for each set were removed as applicable from the constant temperature room (or incubator) at each time point.

Reference substance:

benzoic acid, sodium salt

Remarks:

10.00 μg/L

Test performance:

A preliminary test was conducted to confirm the microbial viability of the natural water to be used in the study and to determine the dose rates, sampling methods, and sampling intervals for the definitive test. See Appendix D for details. Based on the results of the preliminary test, sampling intervals of 0, 7, 14, 28, 43, and 62 days were used for the definitive test.
- Microbial Activity: The reference samples were treated with [14C]benzoic acid at a concentration of 1.00 µg/sample. Duplicate samples were sacrificed after 7 and 14 days of incubation. An average of 74.9% of the applied radiocarbon was converted to 14CO2 (recovered in NaOH traps) after 7 days of incubation, and an average of 84.7% AR of the applied radiocarbon was converted to 14CO2 after 14 days of incubation. This demonstrated the viability of the test system and showed that the microbial activity of the water used in the study was sufficient to conduct the test.

Compartment:

natural water: freshwater

% Total extractable:

46

% CO2:

48.9

% Recovery:

94.8

Remarks on result:

other: Day 62, high dose

Compartment:

natural water: freshwater

% Total extractable:

19.3

% CO2:

72.3

% Recovery:

91.5

Remarks on result:

other: Day 62, low dose

Compartment:

natural water: freshwater

% Total extractable:

99.5

% CO2:

0

% Recovery:

99.4

Remarks on result:

other: Day 62, high dose sterile

Compartment:

natural water: freshwater

% Total extractable:

3.8

% CO2:

85.5

% Recovery:

89.3

Remarks on result:

other: Day 14, reference control

Parent/product:

parent

Compartment:

total system

% Degr.:

62.6

Parameter:

radiochem. meas.

Sampling time:

62 d

Remarks on result:

other: high dose

Parent/product:

parent

Compartment:

total system

% Degr.:

2

Parameter:

radiochem. meas.

Sampling time:

62 d

Remarks on result:

other: high dose sterile

Compartment:

natural water: freshwater

DT50:

24.9 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Transformation products:

not measured

Remarks:

< 10% AR in all sampling interval

Evaporation of parent compound:

not measured

Volatile metabolites:

yes

Residues:

yes

Details on results:

An overview of the results is provided in Table 2 and Table 4 in ‘Any other information on results incl. tables’

- Mass Balance (High Dose): Acceptable mass balance (90 – 110% AR) was achieved in all high dose samples. Individual sample mass ranged 92.0 to 100.2% AR. Average recoveries in high dose water samples decreased from 89.5% AR at time 0 to 31.7% AR at day 28, and represented 44.5% AR at day 62. Average bottle rinse recoveries represented averages of 0.6-1.7% AR throughout the study. Average suspended solid extract recoveries increased from 6.8% AR at time 0 to a maximum of 7.2% AR at day 7, represented 0.3-1.7% for the day 14-43 samplings, and was below detection limits at day 62. Recoveries in the sodium hydroxide traps increased from 10.7% AR at day 7 (the first sampling which included traps) to a maximum average of 57.7% AR at 28 days, and represented 34.9% AR at day 62.
- Mass Balance (Low Dose): Acceptable mass balance was achieved in all low dose samples except for the 7 day replicate A, 14 day replicate B, and both replicates from the 28 day sampling. Individual sample mass balance ranged from 80.6 to 98.1% AR. Average recoveries in low dose water samples decreased from 89.4% AR at time 0 to 18.7% AR at day 62. Average bottle rinse recoveries represented averages of 0.6-2.3% AR throughout the study. Average suspended solid extract recoveries increased from 3.4% AR at time 0 to a maximum of 3.5% AR at day 7, and represented at most 0.7% AR for the remainder of the study. Recoveries in the sodium hydroxide traps represented 19.5% AR at day 7 (the first sampling which included traps) and increased to a maximum average of 67.0% AR at the end of the study (62 days).
- Mass Balance (High Dose Sterile): Acceptable mass balance was achieved in all high dose sterile samples. Individual sample mass balance ranged 95.8 to 103.1% AR. Average recoveries in high dose sterile water samples represented 95.6% AR at day 14, 97.6% AR at day 43, and 98.0% AR at day 62. Average bottle rinse recoveries represented 0.5-1.0% AR throughout the study. Average suspended solid extract recoveries increased from 0.3% AR at day 14 to 2.5% AR at day 43 and represented 1.0% AR at day 62. No volatile traps were used for the high dose sterile samples.
- Product Balance: The product balance was determined by HPLC analysis of sample water layers. All other samples (bottle rinses, suspended solid extracts) represented < 5% AR and were therefore not analyzed during the study. Reference benzoic acid samples were not analyzed.
- Product Balance (High Dose): HPLC analysis of high dose samples throughout the study indicated that the majority of radiocarbon analyzed was [14C]ZBEC. At time 0, [14C]ZBEC represented an average of 89.5% AR in the total system and decreased to an average of 37.4% AR at day 62. Several uncharacterized regions were detected, with the largest representing 11.7% AR in any single sample. However, no uncharacterized peak averaged > 10% AR in any single sampling interval, or greater than 5% AR in consecutive sampling intervals.
- Product Balance (Sterile): HPLC analysis of high dose sterile samples throughout the study indicated that the only peak detected was [14C]ZBEC. [14C]ZBEC represented an average of 95.6% AR after 14 days of incubation and represented an average of 98.0% AR by day 62.
- Proposed Degradation Pathway: The degradation pathway of [14C]ZBEC in natural water under aerobic conditions cannot be elucidated based on the results of this study.
- Kinetic Analysis: Degradation of [14C]ZBEC was significant in surface water. DT50 and DT90 of the degradation of [14C]ZBEC in surface water were calculated using CAKE software version 3.3. The rate of degradation of [14C]ZBEC over 62 days of incubation at 12 ± 2°C was determined to be 24.9 days (SFO model). The statistics and DT50/DT90 values from the kinetics modeling are presented in Table 4 .

Results with reference substance:

- Mass balance (Reference Benzoic Acid): Mass balance was between 80-90% AR for all reference control substance (benzoic acid) samples. Mass balance averaged 85.4 and 89.3% AR for the 7 and 14 day samples, respectively. Average recoveries in reference control substance water samples decreased from 8.6% AR at day 7 to 3.0% AR at day 14. Average bottle rinse recoveries represented 0.4-1.1% AR, and average suspended solid extract recoveries represented 0.4-0.8% AR. Average NaOH trap recoveries increased from 74.9% AR at day 7 to 84.7% at day 14. The recovery in the NaOH traps indicate the viability of the test system.

Table 2a. Mass balance (high dose)

DPM Applied:	246,252
Time Point	Rep	Water	Water Aliquot for Dissolved CO2	Total in Water Layer	Solid Extract	Bottle Rinse	NaOH Traps	Total Recovery %AR1
T0D	A	94.4	NA	94.4	2.6	0.8	NA	97.8
	B	84.5	NA	84.5	11.0	0.7	NA	96.2
Average		89.5	NA	89.5	6.8	0.8	NA	97.0
T7D	A	75.9	NA	75.9	1.7	1.7	12.7	92.0
	B	71.7	NA	71.7	12.6	1.6	8.6	94.5
Average		73.8	NA	73.8	7.2	1.7	10.7	93.3
T14D	A	63.6	19.0	82.6	0.0	1.8	15.8	100.2
	B	44.6	14.2	58.8	0.5	1.5	31.4	92.2
Average		54.1	16.6	70.7	0.3	1.7	23.6	96.2
T28D	A	29.2	5.5	34.7	0.6	0.6	63.1	99.0
	B	34.2	8.1	42.3	0.9	0.8	52.2	96.2
Average		31.7	6.8	38.5	0.8	0.7	57.7	97.6
T43D	A	43.3	13.4	56.7	1.5	0.6	37.5	96.3
	B	39.3	10.9	50.2	1.8	0.6	44.5	97.1
Average		41.3	12.2	53.5	1.7	0.6	41.0	96.7
T62D	A	45.9	14.3	60.2	0.0	1.8	31.5	93.5
	B	43.1	13.6	56.7	0.0	1.1	38.2	96.0
Average		44.5	14.0	58.5	0.0	1.5	34.9	94.8

   

Table 2b. Mass balance (low dose)

DPM Applied:	98,401
Time Point	Rep	Water	Water Aliquot for Dissolved CO2	Total in Water Layer	Solid Extract	Bottle Rinse	NaOH Traps	Total Recovery %AR1
T0D	A	89.7	NA	89.7	2.2	0.7	NA	92.6
	B	89.0	NA	89.0	4.6	0.6	NA	94.2
Average		89.4	NA	89.4	3.4	0.7	NA	93.4
T7D	A	52.4	NA	52.4	4.4	2.1	21.7	80.6
	B	68.4	NA	68.4	2.6	2.2	17.2	90.4
Average		60.4	NA	60.4	3.5	2.2	19.5	85.5
T14D	A	37.8	12.2	50.0	0.0	3.0	40.3	93.3
	B	21.6	5.1	26.7	0.0	1.5	61.7	89.9
Average		29.7	8.7	38.4	0.0	2.3	51.0	91.6
T28D	A	23.4	6.6	30.0	1.3	0.4	56.5	88.2
	B	27.3	8.1	35.4	0.0	0.9	47.5	83.8
Average		25.4	7.4	32.7	0.7	0.7	52.0	86.0
T43D	A	16.6	4.8	21.4	0.0	0.4	76.3	98.1
	B	29.5	7.6	37.1	1.2	0.7	56.9	95.9
Average		23.1	6.2	29.3	0.6	0.6	66.6	97.0
T62D	A	16.1	4.4	20.5	0.0	0.0	70.2	90.7
	B	21.3	6.1	27.4	0.0	1.1	63.8	92.3
Average		18.7	5.3	24.0	0.0	0.6	67.0	91.5

 

Table 2c. Mass balance (sterile high dose)

DPM Applied:	246,252
Time Point	Rep	Water	Water Aliquot for Dissolved CO2	Total in Water Layer	Solid Extract	Bottle Rinse	NaOH Traps	Total Recovery %AR1
T14D	A	96.6	NA	96.6	0.0	0.7	NA	97.3
	B	94.5	NA	94.5	0.5	0.8	NA	95.8
Average		95.6	NA	95.6	0.3	0.8	NA	96.6
T43d	A	95.3	NA	95.3	2.6	1.1	NA	99.0
	B	99.9	NA	99.9	2.4	0.8	NA	103.1
Average		97.6	NA	97.6	2.5	1.0	NA	101.1
T62d	A	98.5	NA	98.5	1.9	0.5	NA	100.9
	B	97.4	NA	97.4	0.0	0.4	NA	97.8
Average		98.0	NA	98.0	1.0	0.5	NA	99.4

Table 2d. Mass balance (Reference control substance)

DPM Applied:	1,035,469
Time Point	Rep	Water	Water Aliquot for Dissolved CO2	Total in Water Layer	Solid Extract	Bottle Rinse	NaOH Traps	Total Recovery %AR1
T7D	A	8.4	NA	8.4	0.8	1.2	74.2	84.6
	B	8.7	NA	8.7	0.8	1.0	75.6	86.1
Average		8.6	NA	8.6	0.8	1.1	74.9	85.4
T14D	A	3.4	0.8	4.2	0.2	0.2	85.2	89.8
	B	2.6	0.8	3.4	0.6	0.5	84.2	88.7
Average		3.0	0.8	3.8	0.4	0.4	84.7	89.3

1Total Recovery = %AR in Water + Extract + Rinse + traps

NaOH = Sodium Hydroxide (caustic trap)

NA: Not Applicable   

Table 3a.  Product balance (high dose)

High Dose (% AR)	Sample	INCUBATION TIME (Days)
		0	7	14		28		43		62
ZBEC	Rep A	94.4	75.9	71.3		0.0		33.3		38.5
	Rep B	84.5	71.7	51.4		7.5		43.5		36.2
	Average	89.5	73.8	61.4		3.8		38.4		37.4
RT 2.80-3.12 min	Rep A	0.0	0.0	1.5		5.2		1.1		2.3
	Rep B	0.0	0.0	2.5		7.7		1.5		1.4
	Average	0.0	0.0	2.0		6.5		1.3		1.9
RT 3.37-3.67 min	Rep A	0.0	0.0	4.7		10.2		2.0		1.3
	Rep B	0.0	0.0	1.7		6.4		1.7		1.5
	Average	0.0	0.0	3.2		8.3		1.9		1.4
RT 21.35-22.15 min	Rep A	0.0	0.0	1.5		7.2		8.3		2.5
	Rep B	0.0	0.0	2.0		11.7		0.0		4.2
	Average	0.0	0.0	1.8		9.5		4.2		3.4
RT 26.88-27.53 min	Rep A	0.0	0.0	0.0		2.6		4.3		6.8
	Rep B	0.0	0.0	0.0		2.4		1.7		4.1
	Average	0.0	0.0	0.0		2.5		3.0		5.5
Others1	Rep A	0.0	0.0	3.7		9.5		7.7		8.8
	Rep B	0.0	0.0	1.2		6.6		1.8		9.4
	Average	0.0	0.0	2.5		8.1		4.8		9.1
Solid Extract	Rep A	2.6	1.7	0.0		0.6		1.5		0.0
	Rep B	11.0	12.6	0.5		0.9		1.8		0.0
	Average	6.8	7.2	0.3		0.8		1.7		0.0
Bottle Rinse	Rep A	0.8	1.7	1.8		0.6		0.6		1.8
	Rep B	0.7	1.6	1.5		0.8		0.6		1.1
	Average	0.8	1.7	1.7		0.7		0.6		1.5
NaOH	Rep A	NA	12.7	15.8		63.1		37.5		31.5
	Rep B	NA	8.6	31.4		52.2		44.5		38.2
	Average	NA	10.7	23.6		57.7		41.0		34.9
Total	Rep A	97.8	92.0	100.3		99.0		96.3		93.5
	Rep B	96.2	94.5	92.2		96.2		97.1		96.1
	Average	97.0	93.3	96.3		97.6		96.7		94.8

 Table 3b.  Product balance (high dose sterile)

HD Sterile (% AR)	Sample	INCUBATION TIME (Days)
		14		43		62
ZBEC	Rep A	96.6		95.3		98.5
	Rep B	94.5		99.9		97.4
	Average	95.6		97.6		98.0
Others1	Rep A	0.0		0.0		0.0
	Rep B	0.0		0.0		0.0
	Average	0.0		0.0		0.0
Solid Extract	Rep A	0.0		2.6		1.9
	Rep B	0.5		2.4		0.0
	Average	0.3		2.5		1.0
Bottle Rinse	Rep A	0.7		1.1		0.5
	Rep B	0.8		0.8		0.4
	Average	0.8		1.0		0.5
Total	Rep A	97.3		99.0		100.9
	Rep B	95.8		103.1		97.8
	Average	96.6		101.1		99.4

1 This column comprises several regions present in the HPLC analysis which do not co-elute with reference standards.

NA: Not Applicable

Table 4. Statistics and DT₅₀/DT₉₀ values from the kinetics modeling

Test Substance	Experimental Set	Sample Phase	DT50 (days)	DT90 (days)	Chi2 Err%	R2	Kinetic Model
[14C]ZBEC	High Dose	Total System	24.9	82.6	28.3	0.5791	SFO
			18.7	262	28.0	0.6412	FOMC
			17.1	682	25.0	0.7677	HS
			15.8	>10,000	29.6	0.6868	DFOP

Validity criteria:

The total recovery (mass balance) at the end of the experiment should be between 90% and 110% for radiolabelled substances.

Observed value:

> 91%

Validity criteria fulfilled:

yes

Conclusions:

In this aerobic mineralization study which followed OECD TG 309, the DT50 of the test substance was determined to be 24.9 days (SFO model). However, after study termination, [14C]ZBEC was found to be unstable to the purification steps used before the study. It has broken down to [14C]dibenzylamine, the species measured during the study. Therefore, the determined DT50 of 24.9 days is for [14C]dibenzylamine.

Executive summary:

 

An aerobic mineralization study was intended to be conducted with [14C]ZBEC in surface water from Tuckahoe Lake, Maryland, USA. The study followed OECD TG 309 and was compliant with GLP criteria. Samples were dosed with ZBEC at two concentrations, 9.86 and 3.92 µg/L. The samples were incubated in the dark on orbital shakers (to provide constant agitation) under aerobic conditions at 12 ± 2 °C for 62 days. In addition, reference and sterile control samples were incubated under the same conditions to confirm the microbial activity of the test water and to examine possible abiotic degradation, respectively. The reference control samples were treated with [14C]benzoic acid at a concentration of 10.00 µg/L, and the sterile control samples were treated with [14C]ZBEC at 9.86 µg/L. The test was performed in flow-through systems which allowed humidified air to pass over the sample headspace and through traps to collect 14C-carbon dioxide (aqueous sodium hydroxide). Sterile samples were not connected to a flow-through system but instead passively aerated with a cotton plug in the neck of the sample bottle. Samples were sacrificed at designated intervals: at time 0 and 7, 14, 28, 43, and 62 days after application. The amount of radioactivity in the water, traps for volatiles, solid extract, and bottle rinse were determined by liquid scintillation counting (LSC), and all water layers were analyzed by high-performance liquid chromatography (HPLC) coupled with flow-through β-ram radio-detector. The samples containing the reference control substance [14C]benzoic acid were sacrificed after 7 and 14 days of incubation. The mineralization of benzoic acid to 14CO2 observed after 14 days of incubation (average of 84.7% AR recovered in NaOH traps) confirmed the microbial activity of the test system. The sterile control samples were sacrificed after 14, 43, and 62 days of incubation to examine possible abiotic degradation.

Acceptable mass balance (90 – 110% AR) was achieved for all samples in all experimental sets except for four low dose samples (one replicate from 7 day, one replicate from 14 day, and both replicates from 28 day samplings), which had recoveries of 80.6-89.9% AR, and the four reference control samples, which had recoveries of 84.6-89.8% AR. As the recovery in the water declined throughout the study, the recovery in NaOH traps increased. The product distribution for all samples analyzed throughout the study indicated that the majority of radiocarbon analyzed was [14C]ZBEC. In the time 0 high dose samples, [14C]ZBEC represented an average of 89.5% AR in the total system and decreased to 37.4% AR at day 62. Several uncharacterized regions were detected in HPLC analysis of the high dose samples, but no individual region exceeded 11.7% AR in any single sample or an average of 9.5% AR at any sampling interval. In the sterile samples, [14C]ZBEC represented an average of 95.6% AR in the total system after 14 days of incubation and an average of 98.0% AR by day 62. Based on the findings, the DT50 for the test substance is determined to be 24.9 days (SFO model). However, after study termination, [14C]ZBEC was found to be unstable to the purification steps used before the study. It has broken down to [14C]dibenzylamine, the species measured during the study. Therefore, the determined DT50 of 24.9 days is for [14C]dibenzylamine. 

Description of key information

The biodegradation in surface water, sediment and soil studies are waived because testings are technically not possible.

Additional information

Based on the ready biodegradability study, it is concluded that the substance is not readily biodegradable. According to column 2 of Annex X, further testing on biodegradation should be considered if the chemical safety assessment indicates the need to further investigate the degradation of the substance and its degradation products. With the available information on the substance, the chemical safety assessment indicates that the risks in water, sediment and soil compartments are controlled, and no further information will have to be provided. 

To conclude on the PBT properties of the substance, an OECD TG 309 followed study was intended to be carried out on [14C]ZBEC. However, upon completion of the 62- day study, ZBEC was found to be unstable in the purification steps and had broken down to [14C]dibenzylamine before applying it to the study. Therefore, the determined DT50 ( = 24.9 days) is for [14C]dibenzylamine instead of [14C]ZBEC. Enormous efforts have been put into overcoming the challenges in purifying and analyzing ZBEC in biodegradation studies. Unfortunately, no promising solution was found. Finally, it is concluded that studying the biodegradation of ZBEC in water, sediment and soil according to OECD standard guidelines is technically infeasible. Therefore, these studies are waived according to the general rules of Section 2 of Annex XI to REACH. The performed study on the breakdown product of [14C]ZBEC (i.e. [14C]dibenzylamine) is included in this endpoint as supporting information. Two expert statements from a professional laboratory testing service are provided as background information to address the detailed information on the experienced challenges and attempted efforts. For the PBT assessment, ZBEC is treated as very persistent (vP) assuming the worst case.