Thiram

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12 June 1985 - 26 September 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

1981

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum: secondary effluent from the aerobic domestic wastewater treatment plant of the city ‘s-Hertogenbosch, Netherlands
- Laboratory culture: No
- Storage length: used at the same day of collection of effluent
- Preparation of inoculum for exposure: The sample was filtered through course filter paper; the first 200 ml were discarded and the remainder of the filtrate was aerated before use as inoculum.
- Pretreatment: No adaptation
- Initial cell/biomass concentration: 1 droplet of filtered and aerated microbial sample per 1 litre

Duration of test (contact time):

28 d

Initial conc.:

2.067 mg/L

Based on:

test mat.

Initial conc.:

10.2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The test substance was dissolved in a mineral nutrient solution providing two different concentrations.
For each determination on the various days separate bottles were incubated. To prepare the test concentrations and controls, bottles with a volume of 3 litres were partially filled with O2-saturated Milli-Q water of 19°C. Subsequently mineral stock solutions following by the test substance were added to reach the appropriate concentrations. Inoculum, 3 dropplets per 3 litres was added if appropriate.
In total six 3 litre bottles following solutions were prepared:
- blank solution
- solution contained reference substance,
- two test solutions at different concentration of test item
- inhibition control
Out of each 3 l bottle 8 Winkler BOD bottles were filled with solutions prepared.
Immediately at the start of experiment (day 0), and at day 5, 15 and 28 dissolved oxygen was determined in all solutions.
Biodegradation (Dt):
Dt is defined as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD). The BOD is expressed as mg O2 per mg test substance.
Toxicity of the test substance:
The toxicity for bacteria in the inoculum was evaluated by comparison of the respective BOD´s of the reference substance, the test substance and the combination of test substance and reference substance.
The test substance should be considered to be toxic to the bacteria present in the inoculum, if the BOD of the combination is less than the sum of the BOD´s of test substance and the reference separately.
Determination of dissolved oxygen:
The concentration of dissolved oxygen was determined with an oxygen electrode (METHROM, Switzerland). Directly before each series of determinations the electrode was calibrated.

Reference substance:

acetic acid, sodium salt

Remarks:

at a concentration of 2.013 mg/L

Parameter:

% degradation (O2 consumption)

Value:

19

Sampling time:

28 d

Remarks on result:

other: 2.067 mg/L

Parameter:

% degradation (O2 consumption)

Value:

40.5

Sampling time:

28 d

Remarks on result:

other: 10.2 mg/L

Details on results:

The test substance reached a biodegradation value of 30% (average biodegradation values of the two tested concentrations) after 28 days. Therefore, as biodegradation remained below the pass level (60%), the test substance is considered as not readily biodegradable.

Results with reference substance:

Under the same conditions sodium acetate (reference substance) was degraded more than 60%.

Table 1: Determination of oxygen concentration and oxygen depletion and evaluation of biodegradation (all values represent a mean value of two measurements, respectively)

	Blank^(a)control	Blank^(b)control	Reference substance			Biodegradation at different TMTD concentrations						Inhibition control(c)
			sodium acetate 2.013 mg/L			2.067 mg /L			10.20 mg /L
Day	mg O2/L	mg O2/L	mg O2/L	O2 dep^(1)	Dt eval^(2)	mg O2/L	O2 dep^(1)	Dt eval^(2)	mg O2/L	O2 dep^(1)	Dt eval^(2)	mg O2/L	O2 dep^(1)
0	9.23	9.29	9.24	-	-	9.25	-	-	9.26	-	-	9.30	-
5	8.98	9.10	7.17	1.88	120	9.05	0.01	0.3	9.19	-0.12	0.0	8.00	1.11
15	8.85	8.83	6.77	2.01	128	8.51	0.28	7.6	5.16	3.64	20.0	7.53	1.31
28	8.85	8.71	6.05	2.61	166	7.97	0.70	19.0	1.33	7.35	40.5	6.96	1.76

^(a): Blank control: Mineral nutrient solution without test item, without inoculum

^(b): Blank control: Mineral nutrient solution without test item, with inoculum

^(c): Inhibition control: Mineral nutrient solution with test item, with inoculum and with reference substance

ThOD (Theoretical oxygen degradation) = 1.78 mg O₂/L

O₂ concentration of the water after aeration and standing before start of test = 9.35 mg O₂/L

⁽¹⁾: O₂dep: depletion (mg BOD/L) after x days::

BOD_d = (m_t0 – m_tx) – (m_b0 – m_bx)

⁽²⁾: D_tevaluation:

D_t = (mg BOD_x/L / (mg TS/L * ThOD)) * 100 (TS: test substance)

Table 2: Validity criteria for OECD 301.

Criterion from the guideline	Outcome	Validity criterion fulfilled
Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.	Difference of extremes of replicate values of the removal of the test chemical were higher than 20% in the high dose level (10.20 mg/L).	no
Percentage degradation of the reference compound reached the pass level by day 14 (≥ 60%).	Percentage degradation of the reference compound was 128% by day 15.	yes
The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.	not calculated	no
Oxygen depletion in the inoculum blank should not exceed 1.5 mg dissolved oxygen/L after 28 days.	The oxygen depletion in the inoculum blank was0.6mg dissolved oxygen/L after 28 days.	yes
The residual concentration of oxygen in the test bottles should not fall below 0.5 mg/L at any time.	The residual concentration of oxygen in one test replicate fell below 0.5 mg/L after 28 days (0.40 mg/L).	no

 

Validity criteria fulfilled:

no

Remarks:

See Table 2 in "Any other information on results incl. tables".

Interpretation of results:

not readily biodegradable

Conclusions:

Due to the presented results, it should be concluded, that TMTD technical is classified as not ready biodegradable using the closed bottle test.
The validity criteria can be considered as fulfilled.

Executive summary:

Materials and methods: The study was designed to assess the ready biodegradability of Tetramethylthiuram disulfide and was conducted according to the OECD guideline 301D.

The test substance was applied in concentrations of 2.067 and 10.20 mg/L, inoculated in a mineral medium and incubated under aerobic conditions in the dark at 20 ± 1 °C.

Degradation was estimated by determination of dissolved oxygen at different intervals.

Results and discussion: The biodegradation of Tetramethylthiuram disulfide at two different concentrations was less than 60% on the experiment termination. The oxygen consumption in the inhibition control containing the test substance at 2.067 mg/L and sodium acetate at 2.013 mg /L was significantly less than it could be expected from the separate values for the test substance and the reference substance.

Tetramethylthiuram disulfide showed a toxic impact on the inoculated bacteria when exposed to 2mg/L test material.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Version / remarks:

2006

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3120 (Sealed Vessel Carbon Dioxide Production Test)

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Remarks:

The department of health of the government of the United Kingdom

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Obtained from the aeration tank of Worlingworth sewage treatment works (Church road, Worlingworth, Suffolk, IP13 7NU, UK), which treats predominantly domestic waste.
- Preparation of inoculum for exposure: Each culture bottle was inoculated and a magnetic stirring bar was added. The vessels were then placed on an electrically-operated magnetic stirrer and aerated until the day of test initiation (Day 0) with a supply of air that
had been treated to remove carbon dioxide by passing it through cylinders containing fused calcium chloride and Carbosorb AS (Soda lime made by VWR AnalaR Normapur grade).
- Pretreatment: On the day of collection, the sample was passed through a sieve with a mesh of ca. 1 mm². A sub-sample (ca. 250 mL) was removed and centrifuged at ca. 3000 rpm for ca. 1 minute and the supernatant removed. The sample was then made up to volume with mineral salts medium (MSM) and centrifuged. This procedure was repeated twice and the sample was maintained aerated until required.
- Water filtered: no

Duration of test (contact time):

28 d

Initial conc.:

4.9 other: mg C/L

Based on:

TOC

Parameter followed for biodegradation estimation:

inorg. C analysis

Details on study design:

TEST CONDITIONS
- Composition of medium: As described in OECD 310 (2006)
- Solubilising agent: An aliquot of test substance dissolved in acetone (0.1 mL) was added to the designated test vessels, after which the acetone was evaporated off using a steady stream of nitrogen to leave a residue of the test substance on the vessel walls. The vessels were left to stand for 1 hour, and then flushed further with nitrogen, after which minimum salt medium (100 mL) was added.
- Test temperature: 19 to 21 °C
- pH adjusted: Yes. To 7.4 ± 0.2 with 5M HCl
- Suspended solids concentration: 0.4 mg/L

TEST SYSTEM
- Culturing apparatus: Sealed vessels (160 mL)
- Number of culture flasks/concentration: 26 with test substance (inoculated mineral salts medium plus test substance Thiram, 4.9 mgC/L)
- Method used to create aerobic conditions: Test vessels were aerated until the day of test initiation (Day 0) with a supply of air thathad been treated to remove carbon dioxide.
- Measuring equipment: The total inorganic carbon (TIC) content of each culture was determined using an OI Analytical Model 1010 Total Organic Carbon Analyser.
- Test performed in open system: No, the vials were immediately sealed with teflon-coated butyl rubber septa and aluminium crimp seals.
- Details of trap for CO2 and volatile organics if used: Concentrated NaOH was added to the test cultures and shaken. Gaseous CO2 evolved during biodegradation was trapped as carbonate or other forms of inorganic carbon in the liquid phase.

SAMPLING
- Sampling frequency: Days 0, 5, 11, 14, 18, 21 and 28
- Sampling method: On each sampling occasion, the appropriate number of vials were injected with 1 mL of sodium hydroxide (nominally 7M) and shaken for at least one hour. The total inorganic carbon content (TIC) of each sample was then determined using a carbon analyser. TIC analysis was conducted in duplicate on three cultures from each group on each sampling occasion except on the last day of the test, when five cultures were analysed.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Blank-controls contained inoculated minimum salt medium alone, 0.4 mg solids/L (26 flasks).
- Positive control: Inoculated mineral salts medium plus sodium benzoate, 4.9 mgC/L (17 flasks)
- Toxicity control: The inhibition assay contained a mixture of the reference substance and test substance (both 4.9 mgC/L) in pre-aerated minimum salt medium labelled inhibition (9 flasks).
- Water blanks: Ultrapure water blanks for TIC and TOC analysis (8 flasks)

Reference substance:

benzoic acid, sodium salt

Remarks:

at a nominal concentration of 4.9 mg C/L

Preliminary study:

The results of the preliminary investigation showed that there was unlikely to be any abiotic production of CO2 with the addition of 7M NaOH used on study. The chemistry results were acceptable (101% of nominal) and the total inorganic carbon (TIC) results observed were consistent between test and control vessels.

Test performance:

The pH of the test/control and reference minimum salt medium at the start of the test was 7.52. The temperature of a 100 mL volume of water held under test conditions ranged from 19.8 °C to 20.5 °C during the test period.

Parameter:

% degradation (inorg. C analysis)

Value:

30

Sampling time:

28 d

Details on results:

Production of CO2 by mixtures containing Thiram was equivalent to 11.2% of the theoretical maximum after approximately Day 11, which increased (13.8, 18.0 and 23.6% by Days 14, 18 and 21, respectively) to give a degradation of 30.0% by the end of the test on Day 28.

Results with reference substance:

Sodium benzoate alone had been biodegraded by 60.5% on Day 5, by 84.0% on Day 14, and by 85.9% on Day 28. Sodium benzoate had been degraded by 62.0% after 5 days in the presence of Thiram, which confirmed that Thiram was not inhibitory to the activity of the microbial inoculum.

For details on inorganic carbon measurement results and percentage biodegradation see attachment.

Table 1: Validity criteria for OECD 310

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mean percentage degradation in vessels containing the reference substance is >60% by the 14th day of incubation	The reference substance sodium benzoate alone had been biodegraded by 84.0% on Day 14.	yes
The mean amount of TIC present in the blank controls at the end of the test is <3 mg C/L	The mean inorganic carbon concentration from duplicate samples of fife cultures ranged from 0.853 to 1.074 mg IC/L.	yes

 

Validity criteria fulfilled:

yes

Remarks:

See Table 1 in "any other information on results incl. tables".

Interpretation of results:

not readily biodegradable

Description of key information

Tetramethylthiuram disulfide (CAS No. 137-26-8) is not readily biodegradable: 30% (inorg. C analysis) in 28 days (OECD 310)

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

One key study (2014) investigating the potential for ready biodegradability of tetramethylthiuram disulfide (CAS No. 137-26-8) is available. This test was conducted according to the OECD Guideline No. 310, EPA Guideline OPPTS 835.3120 and under GLP conditions. Mineral salts medium was inoculated with non-adapted activated sludge and exposed to a test substance concentration of 4.9 mg C/L for 28 d. A toxicity control was run in parallel to assess the potential inhibitory effect of the test substance. After 28 days, 30% of the test substance were degraded. Thus, as tetramethylthiuram disulfide biodegradation did not reach the pass level of 60%, it can be considered as not readily biodegradable. The toxicity control showed 62.0% degradation of the reference substance sodium benzoate after 5 days, thus no inhibitory effects of Thiram towards the inoculated bacteria can be assumed.

 

This result is supported by another study (1985) performed according to OECD 301D and GLP. Non-adapted activated sludge was exposed to test substance concentrations of 2 mg/L and 10.2 mg/L. After 28 days, the test compound reached a biodegradation value of 30%.

 

In conclusion tetramethylthiuram disulfide (CAS No. 137-26-8) is not readily biodegradable.