2-hydroxy-4-(methylthio)butyronitrile

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:

From 2/07/1998 (Study initiation) to 13/08/1998 (Study termination)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to the EU guideline and the GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-4-(methylthio)butyronitrile

EC Number:

241-756-3

EC Name:

2-hydroxy-4-(methylthio)butyronitrile

Cas Number:

17773-41-0

Molecular formula:

C5H9NOS

IUPAC Name:

2-hydroxy-4-(methylsulfanyl)butanenitrile

Constituent 2

Reference substance name:

2-hydroxy-4-(methylsulfanyl)butanitrile

IUPAC Name:

2-hydroxy-4-(methylsulfanyl)butanitrile

Details on test material:

See field "Confidential details on test material"

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge : microorganisms from a domestic sewage treatment plant (i.e. "station de traitement biologique urbaine de Saint Germain au Mont d'Or"), sample taken the 2 July 1998
- Preparation of inoculum for exposure: inoculum has been prepared by filtration on a glass fiber pre-filter (removal of big particles), centrifugation of the eluent and washing. The bacterial centrifugation pellet has been taken up in a Ringer's solution 1/4. The inoculum has been used within 24 hours after its preparation.
- Initial cell/biomass concentration: The concentration in microorganisms of the inoculum, measured by optical density and subsequently checked by count in petri dishes, was 3x10E8 bacteries/mL. Inoculum has been used at 1% in the test medium.

Duration of test (contact time):

42 d

Initial test substance concentration

Initial conc.:

20 other: mg/kg

Based on:

other: Total Carbon

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium:
85 mg of KH2PO4
217.5 mg of K2HPO4
5 mg of NH4Cl
334 mg of Na2HPO4, 2 H2O
22.5 mg of MgSO4, 7 H2O
36.4 mg of CaCl2, 2 H2O
0.25 mg of FeCl3, 6 H2O
1000 g of bidistilled water.
- Additional substrate: none
- Solubilising agent : none
- Test temperature: 21.4 - 22.5 °C
- pH: no data
- pH adjusted: no data
- CEC (meq/100 g): no data
- Aeration of dilution water: no data
- Suspended solids concentration: no data
- Continuous darkness: yes
- Other: bistilled water (DOC < 4 mg/kg) has been used for the preparation of the test solutions


TEST SYSTEM
- Culturing apparatus: 500 mL glass Erlenmeyer flasks vacuum-tight sealed, containing 200g of test medium.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: no data
- Measuring equipment: DOC has been measured by a Dohrmann Carbon Analyser DC 190 with oxidation at 900°C, at days 0, 4, 7, 14, 21, 28, 35 and 42.
- Test performed in closed vessels due to significant volatility of test substance: yes
- Other: culturing apparatus and all materials were previously washed to remove organic mater and toxic substances for microorganisms (Treatment by Decon)


SAMPLING
- Sampling frequency: DOC has been measured at days 0, 4, 7, 14, 21, 28, 35 and 42.
- Sampling method: in order to compensate any evaporation losses from the flask, before sampling, the quantity of the test medium of each flask has been checked by weight, and, if needed, the weight has been adjusted to the weight measured after the previous sampling by bidistilled water. Samples were centrifugated ( 6000 rpm at 6°C during 20 minutes). The COD content has been measured in supernant.
- Sample storage before analysis: no data.


CONTROL AND BLANK SYSTEM
- Inoculum blank: two flasks for blank control (inoculum at 1% in the test medium)
- Abiotic sterile control: one flask containing the test substance and 1% of Na Cl3 at 10g/L.
- Toxicity control: one flask containing the inoculum, the test substance (20 mgOC/kg) and the reference substance (20 mgOC/kg)


CALCULATION METHODS:
In the test flasks, the DOC content has been calculated as follows:
DOCt = DOCm(t) - (DOCb1(t) + DOCb2(t))/2
with:
DOCt = Dissolved Organic Carbon content at time t (mg/kg)
DOCm(t) = Dissolved Organic Carbon measured (mg/kg) in the test flasks (test substance, reference substance and toxicity control)
DOCb1(t) = Dissolved Organic Carbon measured (mg/kg) in the blanK flask 1 (containing only inoculum) at time t
DOCb2(t) = Dissolved Organic Carbon measured (mg/kg) in the blanK flask 2 (containing only inoculum) at time t

In the abiotic control flask, the DOC content has been determined as follows:
DOCt = CODa(t)
with
DOCt = Dissolved Organic Carbon content at time t (mg/kg)
CODa(t) = Dissolved organic carbon measured (mg/kg) in the abiotic control flask at time t

Biodegradation calculation:
The rate of biodegradation has been calculated as follows:
% degradation = [{(C0 - Cb0) - (Ct - Cbt)} / (C0 - Cb0)] x 100
with:
C0 = starting DOC content in the test flasks (test substance, reference substance and toxicity control)
Ct = DOC content at time t in the test flasks (test substance, reference substance and toxicity control)DOCm(t) = DOC content
Cb0 = starting mean DOC content in the blank flasks 1 and 2
Cbt = mean DOC content at time t in the blank flasks 1 and 2

Abiotic degradation calculation
The percent of abiotic degradation has been calculated as follows:
% abiotic degradation = (C0 - Ct) / C0 x 100
with:
C0 = starting DOC content in the abiotic control flask
Ct = DOC content at time t in the abiotic control flask

Reference substance

Reference substance:

acetic acid, sodium salt

Remarks:

at 20 mgOC/kg

Results and discussion

Preliminary study:

No data.

Test performance:

No unusual observation reported.

% Degradation

% Degradation 1

Parameter:

% degradation (DOC removal)

Value:

11

Sampling time:

7 d

Remarks on result:

other: Mean of measurement in two replicates (i.e. 12% and 10%)

% Degradation 2

Parameter:

% degradation (DOC removal)

Value:

25

Sampling time:

14 d

Remarks on result:

other: Mean of measurement in two replicates (i.e. 34% and 16%)

% Degradation 3

Parameter:

% degradation (DOC removal)

Value:

57.5

Sampling time:

21 d

Remarks on result:

other: Mean of measurement in two replicates (i.e. 71% and 44%)

% Degradation 4

Parameter:

% degradation (DOC removal)

Value:

63.5

Sampling time:

28 d

Remarks on result:

other: Mean of measurement in two replicates (i.e. 65% and 62%)

% Degradation 5

Parameter:

% degradation (DOC removal)

Value:

87.5

Sampling time:

42 d

Remarks on result:

other: Mean of measurement in two replicates (i.e. 89% and 86%)

Details on results:

The measured BOD concentrations are reported in table 1.
The calculated biodegradation rates are reported in table 2.

BOD5 / COD results

Results with reference substance:

The measured BOD concentrations are reported in table 1.
The calculated biodegradation rates are reported in table 2.

Any other information on results incl. tables

Table 1: Dissolved Organic Carbon measurements

Sampling time	Blank control (flask 1)	Blank control (flask 2)	Test substance (flask 1)	Test substance (flask 2)	Reference substance	Toxicity Control	Abiotic degradation control
0	1.6	2.2	22	22.1	26.2	43.1	20.2
4	1.9	1.5	19.3	19	5.3	17.1	19
7	2.1	1.7	21.9	20.1	4.2	9.1	24.3
14	2.5	1.7	15.3	19.1	3.9	8.2	26.1
21	1.3	1	7	12.5	2.1	7.3	16.3
28	1.3	1.1	8.3	8.9	2.9	7.1	17.3
35	1.5	1.2	4.2	5.1	2.4	4.5	20
42	1.5	1.6	3.8	4.3	2.6	4.6	19.4

Table 2: biodegradation rates

Sampling time	Test substance (flask 1)	Test substance (flask 2)	Reference substance	Toxicity Control	Abiotic degradation control
4	12	13	85	63	6
7	12	10	90	83	0
14	34	16	93	85	0
21	71	44	96	85	19
28	65	62	93	86	14
35	86	81	96	92	0
42	89	86	96	93	4

Validity criteria

- Difference between extremes values of replicates of test substance at day 28 and at the end of the incubation period are below 20%

- Percent of degradation of the reference substance is > 60% within 14d

- In the inhibition control (test sub + ref sub) the degradation of the reference substance is > 25% within 14d (there is no inhibition of the degradation of the reference substance by the test substance).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

(validity criteria of the OECD TG)

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

In the test conditions, HMTBN showed 63.5% and 87.5 % of biodegradation after 28 and 42 days respectively. HMTBN reached the pass level for ready biodegradability but failed the 10-day window. Therefore, it should not be considered as readily biodegradable.

Executive summary:

The ready biodegradability of HMTBN was tested under GLP conditions in a DOC-Die-Away-Test according to the EU Directive 92/69/EEC C.4 -A. Activated sludge from a domestic sewage treatment plant served as inoculum. The samples were incubated for 42 days, in duplicate for the blank controls and the test item. DOC was analyzed after 4, 7, 14, 21, 28, 35 and 42 days. After 28 days, the test substance was degraded at 63.5% and after 42 days at 87.5 % (based on DOC removal). HMTBN reached the pass level for ready biodegradability but failed the 10-day window. Therefore, it should not be considered as readily biodegradable.

The control of the inoculum has been performed using sodium acetate as reference substance. It showed that the inoculum was suitable for the study. The toxicity control showed that HMTBN was not toxic to the inoculum and the abiotic degradation control showed no abiotic degradation. The OECD validity criteria were fulfiled.