(R*,R*)-1,4-dimercaptobutane-2,3-diol

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 March 2020 - 23 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(R*,R*)-1,4-dimercaptobutane-2,3-diol

EC Number:

222-468-7

EC Name:

(R*,R*)-1,4-dimercaptobutane-2,3-diol

Cas Number:

3483-12-3

Molecular formula:

C4H10O2S2

IUPAC Name:

1,4-disulfanylbutane-2,3-diol

Specific details on test material used for the study:

Lot no. 44606300 / 50774
Test item storage 2–8 °C, under nitrogen

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source: The activated sludge was sampled from the aeration tank of the ARA Werdhölzli (CH-8048 Zürich), a municipal biological wastewater treatment plant, on 5 March 2020, 8:30 a.m.

- Preparation of inoculum for exposure: In accordance with the guideline, this sludge was not pre-adapted to the test item but pre-conditioned to reduce the amount of O2 consumed in the blank controls. In the pre-conditioning procedure, the sludge was washed twice with tap water and once with test medium right after sampling from the wastewater treatment plant. After centrifugation, the sludge was suspended in test medium at 3–5 g/l dry matter and kept under constant aeration and stirring without feeding for five days, until test start. Before the start of the test, the actual dry matter content of this suspension was determined.

- Pretreatment: In accordance with the guideline, this sludge was not pre-adapted to the test item.

- Concentration of sludge: On the day of the test start, an appropriate aliquot of this sludge suspension was first homogenised and subsequently diluted to 300 mg/l dry matter (i.e. ten times higher than the final test concentration of 30 mg/l, as it will be further diluted by a factor of ten during the test set-up).

Duration of test (contact time):

43 d

Initial test substance concentration

Initial conc.:

64.3 mg/L

Based on:

other:

Remarks:

nominal concentration: The test item was applied by stock solution in flasks T (Test) and X (Toxicity control) and by direct addition in flask A (Abiotic sterile control).

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
Test medium
A mineral salt medium was prepared according to the test guideline and used for sludge conditioning and testing (except for the abiotic sterile control and the water flask):
* mineral stock solution A: 8.5 g/L KH2PO4, 28.49 g/L K2HPO4.3H2O, 33.4 g/L Na2HPO4.2H2O, 0.5
0 g/L NH4Cl
* mineral stock solution B: 36.4 g/L CaCl2.2H2O
* mineral stock solution C: 22.5 g/L MgSO4.7H2O
* mineral stock solution D: 0.25 g/L FeCl3.6H2O
* Final test medium: 10 mL of solution A and 1 mL of solutions B, C and D per L of test medium

- pH: The pH value of the test medium (7.4±0.2 according to the guideline) was adjusted to 7.2, as the test item is known to undergo a pH-dependent oxidation in the presence of oxygen. This oxidation proceeds faster the higher the pH.
- Incubation: At 22±2 °C in a temperature-controlled dark room.
- Feed: None, test and/or reference item as sole organic carbon sources.
- Test duration: 43 days

TEST SYSTEM
- Test units 2.3 l closed glass bottle containing a total volume of test suspension of 2 l; aerated with CO2-free air and fitted to gas-absorption bottles containing 140 ml of 0.13 M potassium hydroxide (KOH).
- Test performed in triplicate (three test flasks): The test item was applied by stock solution (test item in test medium).

CONTROL AND BLANK SYSTEM
- Blank control (B) (number of replicates: 3), containing: Inoculum, test medium
- Procedure control (P) (number of replicates: 3), containing: Inoculum, test medium, reference item (Replicate 1: 34.1 mg/l; Replicate 2: 34.0 mg/l; Replicate 3: 34.0 mg/l; corresponding to 19.9; 19.8, and 19.8 mg TOC/l, respectively)
- Test (T) (number of replicates: 3), containing: Inoculum, test medium, test item (Replicates 1, 2 & 3: 64.3 mg/l; corresponding to 20.0 mg TOC/l)
- Toxicity control (X) (number of replicates: 1), containing: Inoculum, test medium, test item (64.3 mg/l), reference item (34.0 mg/l); corresponding to 39.8 mg TOC/l
- Abiotic sterile control (A) (number of replicates: 1), containing: Ultra-pure water, test item (64.3 mg/l; corresponding to 20.0 mg TOC/l), 0.04 mM HgCl2 (sterilising agent to prevent microbial decomposition)

The test item was applied by stock solution in flasks T and X and by direct addition in flask A. The reference item was applied by direct addition in flasks P and X. The pH value in the stock solutions was adjusted, if necessary, to be at the same level of 7.2 as in the blank control. The pH in flask X was checked and adjusted to 7.2, if necessary. In flasks A and W, the pH is not relevant, as it contains only ultra-pure water as solvent and thus no relevant buffering capacity.

SAMPLING
Samples were taken at test start from the test medium and the sludge suspension for the measurement of the inorganic carbon (IC) in the test suspension and from the test item stock solution for the measurement of the dissolved organic carbon (DOC).
The gas-absorption bottles were filled with the 0.13 M KOH solution. The treatment flasks were connected to the air supply (CO2-free, synthetic) and the gas-absorption bottles were connected to the test flasks, so that the air leaving the individual vessels is passed through the KOH solution and the CO2 produced by mineralisation of the test item is trapped therein.
All treatment flasks were stirred thoroughly and aerated with the air supply mentioned above for the whole test period.
The CO2 trapped in the KOH solutions was determined as inorganic carbon at frequent time intervals to allow for the assessment of the 10-day window for biodegradation in the vessels. The temperature was monitored by regular readings of a datalogger.

PROLONGATION OF THE STUDY
After 28 days, at which timepoint an OECD 301 B study is typically stopped, it was decided together with the sponsor to prolong the study, as the three test replicates (T) showed strongly diverging degradation patterns.
To determine the ideal timepoint to stop this study, the measurements, which normally take place only at the end of the test (determination of pH as well as IC and DOC concentrations in the treatment flasks, if applicable) were performed on samples taken on days 28 and 35 in addition to further samplings of the CO2 trapped in the KOH solutions. It was subsequently decided by the sponsor to stop the study after 43 days of duration, as the second test replicate seemed to reach a plateau comparable to the one of the first test replicate. Therefore, determinations of pH as well as IC and DOC concentrations in the treatment flasks were then performed for the last time together with CO2 determination in the KOH flasks.

STATISTICAL METHODS
No statistical analysis was performed.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

% Degradation 1

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

Replicate 1

Value:

53

Sampling time:

43 d

% Degradation 2

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

Replicate 2

Value:

62

Sampling time:

43 d

% Degradation 3

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

Replicate 3

Value:

34

Sampling time:

43 d

Details on results:

The biodegradability of 1,4-Dithiothreitol was calculated to be 53%, 62% and 34% in the first, second and third test replicate, respectively, after an incubation time of 43 days, based on CO2 evolution and expressed as % of the total organic carbon (TOC) applied.
The observed biodegradation of the test item was preceded by a marked lag-phase (time to 10% degradation), which strongly differed in length among replicates (i.e. 15, 26 and 37 days for the first, second and third replicate, respectively).
At the end of the 10-day window, the biodegradation of 1,4-Dithiothreitol reached 42% and 56% in the first and the second test replicate. The third test replicate did not reach the end of the 10-day window by test end.
The total elimination calculated based on the DOC measurements performed after 28, 35 and 43 days follows closely the biodegradation determined for each test replicate. The first replicate being the fastest and levelling off at 56% to 58% elimination after about 28 days, which is mirrored by the second replicate but with a delay of about seven days, whereas the third replicate starts even later and reaches about 47% elimination after 43 days.

BOD5 / COD results

Results with reference substance:

The procedure control with sodium benzoate reached a biodegradation of 87% after 14 days, thus confirming suitability of inoculum and test conditions. The elimination determined for the sodium benzoate replicates ranged from 94 to 99% at test end.

Any other information on results incl. tables

Toxicity control

According to the OECD guideline 301 a substance is considered having inhibitory (i.e. toxic) effects on the inoculum if less than 25% degradation after 14 days are observed in the toxicity control. Since the biodegradation exceeded this pass level, it can be concluded that the test item does not have any significant toxic effects on the microbial population at the applied initial test concentration of 64.3 mg/l.

In addition, the toxicity control also seems to show the delayed onset of the test item degradation, as the curve continues to increase from day 14 on, whereas the curve of the procedure control starts to level off at this timepoint.

Abiotic sterile control

No significant degradation of the test item based on CO2 development was observed in the abiotic sterile control in the absence of microorganisms.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

1,4-Dithiothreitol (CAS no. 3483-12-3) did not reach the pass level of 60% biodegradation in the CO2 evolution test within the 10-day window and therefore, cannot be termed as readily biodegradable.

Executive summary:

The biodegradability of 1,4-Dithiothreitol (CAS no. 3483-12-3) exposed to microorganisms derived from activated sludge of a municipal sewage treatment plant was investigated under aerobic static exposure conditions, following the OECD guideline 301 B. The biodegradation was assessed based on CO2 evolution and expressed as % of the total organic carbon (TOC) applied.

The biodegradability of 1,4-Dithiothreitol was calculated to be 53%, 62% and 34% in the first, second and third test replicate, respectively, after an incubation time of 43 days. The observed biodegra-dation was preceded by a marked lag-phase, which strongly differed between replicates (i.e. 15, 26 and 37 days for the first, second and third replicate, respectively).

The total elimination calculated based on the DOC measurements performed after 28, 35 and 43 days follows closely the biodegradation determined for each test replicate. The first replicate being the fastest and levelling off at 56 to 58% elimination after about 28 days, which is mirrored by the second replicate but with a delay of about seven days, whereas the third replicate starts even later and reaches about 47% elimination after 43 days (see Table 4).

The procedure control with sodium benzoate reached a biodegradation of 87% after 14 days, thus confirming suitability of inoculum and test conditions. The elimination determined for the sodium benzoate replicates ranges from 94 to 99% at test end.

1,4-Dithiothreitol (CAS no. 3483-12-3) did not reach the pass level of 60% biodegradation in the CO2 evolution test within the 10-day window and therefore, cannot be termed as readily biodegradable.

All validity criteria were fulfilled.