Iodomethane

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:

22 June 2011 to 20 July 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: The activated sludge used as microbial inoculum was from the local municipal wastewater treatment plant in Romanshorn Switzerland
- Laboratory culture: Yes
- Collection date: 21 June 2011
- Preparation of inoculum for exposure: Sludge was concentrated by centrifugation at 1000 rpm for 5 minutes. The supernatant was discarded. The sludge was washed 3 times with tap water and the supernatant was discarded after centrifugation. The moisture content of the activated sludge was determined using a drying cabinet.
- Concentration of sludge: A sludge solution containing 3.0 g solids/L tap water was prepared by dissolving 356.918 g wet sludge on a total volume of 3000 mL one day prior to test initiation.
- The inoculum was fed with 150mL synthetic sewage feed for the precondition phase and was stored over night in an environmental chamber under aeration at 20.0 to 21.0 °C.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Initial conc.:

39.5 mg/L

Based on:

ThOD/L

Initial conc.:

250 mg/L

Based on:

test mat.

Initial conc.:

98.6 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium was prepared by dissolving appropriate amounts of the stock solutions in deionised water. One batch of deionised water was used. The dissolved organic carbon (DOC) of this batch was determined to be 0.23 mg/L, which represents less than 10 % of the DOC added over the test material.
Solution # A: KH2PO4 0.850 g, K2HPO4 2.181 g, Na2HPO4.2 H2O 3.346 g, NH4Cl 0.053 g and 100 mL Reagent Grade Water.
Solution # B: CaCl2•2H2O 3.646 g and 100 mL Reagent Grade Water.
Solution # C: MgSO4•7H2O 2.258 g and 100 mL Reagent Grade Water.
Solution # D: FeCl3•6H2O 0.027 g and 100 mL Reagent Grade Water.
- Due to the low water solubility of the test material the application was performed by direct application into the test vessels. The test material was applied using a syringe with μL scale. Prior to application, the density of the test material was evaluated to calculate the volume required for application. Based on the evaluated density of 2.02 g/mL the application volume of the test material was calculated to be 8.1 μL for 39.5 mg ThOD/L and 20.3 μL for the 98.6 mg ThOD/L, for the low and high application rate, respectively.
- Sterilising Agent: A sterilising agent HgCl2 from Sigma Aldrich, Cat. 21,546-5, Lot # 010M1509 was used. For application, an amount of 0.0205 g was directly added to the test solution, resulting in a final HgCl2 concentration of 0.125 g/L. The pH of the abiotic sterile control was determined to be 7.05 after addition of the sterilising agent. The pH was thereafter adjusted to 7.40 with one drop of 2 molar KOH solution.
- Test temperature: 21.7 to 23.6 °C
- pH: The pH at test start was 7.45 in the inoculum blank suspension. The pH of the abiotic sterile control was adjusted from 7.05 to 7.40 with one drop of 2M KOH.
- After set up of the test systems, the test flasks were closed air tight with the OxiTop® measuring heads equipped with sodium hydroxide pellets as CO2 absorber.
- Suspended solids concentration: 3.0 g/L
- Synthetic Sewage Feed: The synthetic sewage feed was prepared under a similar study with Smithers Viscient AG study number # 1121.015.790. The synthetic sewage feed was prepared in deionised water and contained: Peptone 8.0 g, Meat Extract 5.5 g, Urea 1.5 g, NaCl 0.35 g, CaCl2.2H2O 0.2 g, MgSO4.7H2O 0.1 g and K2HPO4 1.4 g. The deionised water was analysed for its non-purgable dissolved organic carbon content. The content was determined to be lower than 1 mg/L.

TEST SYSTEM
- Culturing apparatus: The test was performed using OxiTop OC 110 systems which were placed on a inductive stirring system (WTW OxiTop IS 6-Var) in a WTW Thermostat cabinet. The used software was Achat OC, Version 2.03. The measuring head from OxiTop® was air-tight fixed on the 500 mL brown glass flask with a holder for the sodium hydroxide pellets (to absorb the produced CO2) in the bottle neck. The test vessels contained 164 mL of test solution.
- Number of culture flasks/concentration: 2

MONITORING
- The temperature of the environmental chamber was recorded daily from day 2 onwards throughout the exposure period using a min/max thermometer and was maintained at a temperature of 22 ± 1 °C.
- The pressure in the test flasks was recorded at regular intervals throughout the study by means of the OxiTop® measuring heads. Data about the change in pressure in the test vessels were recorded every 112 minutes over 28 days. After experimental termination, the data was transferred to the OxiTop® controller OC110 and from there it was transferred to a computer to further evaluate the data.

CONTROL AND BLANK SYSTEM
- Inoculum blank: containing only inoculum and mineral medium (n=2).
- Abiotic sterile control: containing test material, sterilising agent (HgCl2) and mineral medium (n=1).
- Toxicity control: containing test material (100 mg/L), reference material (100 mg/L), mineral medium and inoculum (n=1).
- Toxicity control high: containing test material (250 mg/L), reference material (100 mg/L), mineral medium and inoculum (n=1).
- Procedure control: A stock solution was prepared by dissolving 0.1643 g of sodium benzoate in 25 mL mineral medium. The addition of 2.5 mL of the reference stock solution resulted in a definitive concentration of 100 mg reference material/L which was equivalent to 166.5 mg ThOD/L. The ThOD of 1.67 mg oxygen/mg reference material was used for the calculation (n=1).

CALCULATIONS
- Test material:
Molecular formula of test material: CH3I , molecular weight: 141.94 g/mol.
ThOD = 16*[2C+0.5(H-Cl-3N)+3S+2.5P+0.5Na-O]/MW
ThOD = 16*[(2*1)+0.5(3-0-0)+3*0+2.5*0+0-0]/ 141.94
ThOD = 0.39 mg O2/mg test material

- Reference material (sodium benzoate):
Molecular formula of test material: C7H5NaO2, molecular weight: 144.11g.
ThOD = 1.67 mg O2/mg reference material

- Biological Oxygen Demand BOD: Degradation of the test and reference material was calculated using the BOD.
The Biological Oxygen Demand BOD (mg O2/L) was calculated with following equation:
BOD = [M(O2)/(R.Tm)].[((Vges-VF1)/VF1) + α(TM/T0)].Δp(O2)
where:
M(O2) = Molecular weight (32000 mg/mol)
R = Gas constant (83.144 mbar/mol K)
T0 = Reference temperature (273.15 K)
Tm = Measure temperature
Vges = Flask volume (nominal volume in mL)
VF1 = Sample volume in mL
α = Bunsenscher absorptions coefficient (0.03103)
Δр(O2) = Oxygen partial pressure difference (mbar)

- The percent degradation was calculated according to the following equation:
% Biodegradation = [(BODtest material/reference(mg O2/L) –BODinoculum(mg O2/L)) / ThODtest material/reference(mg O2/L)] x100

Reference substance:

benzoic acid, sodium salt

Test performance:

The validity criteria were met

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

VALIDITY CRITERIA
- According to the validity criteria the BOD in the inoculum blanks should not exceed 60 mg/L medium at the end of the test. During this study, the BOD from inoculum blanks replicates A and B were 78.7 and 78.7 mg O2/L at day 28, respectively. This validity criterion was therefore not fulfilled. Details about this validity criterion are given in section 5.0. The difference between the blank control replicates was not exceeding 5 % at any time point during the study. This validity criterion is accepted.
- The average BOD at test day 28 measured in the two test vessels with the test material (low concentration) was 67.9 mg O2/L. The degradation was calculated to be -27.6 % for test suspension at the end of the test. The difference between the test suspension replicates was less than 20 %. The average BOD at test day 28 measured in the two test vessels with the high concentration of the test material was 65.2 mg O2/L. The degradation was calculated to be -13.8 % for this test suspension at the end of the test.
- The BOD measured in the procedure control up to day 28 was 230.8 mg O2/L which corresponds to 91.4 % degradation of the reference material. After 2 days of incubation, the degradation already reached a level of 64.4 % degradation. The validity criterion regarding the degradation of the reference material is therefore fulfilled. This rapid degradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.
- The BOD in the toxicity control (low concentration of test material) was compared with the procedure control. The BOD in the toxicity control was determined to be 154.8 mg O2/L up to day 14 and 187.4 mg O2/L at day 28, which corresponds to 49.8 and 65.3 % degradation, respectively. Since more than 25 % degradation was observed, the test material was not inhibitory to the inoculum.
-The BOD in the toxicity control with the high concentration of test material was compared with the procedure control. The BOD in the toxicity control was determined to be 171.1 mg O2/L up to day 14 and 190.1 mg O2/L at day 28, which corresponds to 59.5 and 66.9 % degradation, respectively. Since more than 25 % degradation was observed, the test material was not inhibitory to the inoculum, at a concentration of 250 mg/L.
- The abiotic sterile control did not show degradation of the test material.

Results with reference substance:

- The BOD measured in the procedure control up to day 28 was 230.8 mg O2/L which corresponds to 91.4 % degradation of the reference item. After 2 days of incubation, the degradation already reached a level of 64.4 % degradation. The validity criterion regarding the degradation of the reference item is therefore fulfilled. This rapid degradation of sodium benzoate confirmed the presence of an active microbial population and system integrity.

Table 1: Calculated % Biodegradation

Day of Measurement	Calculated biodegradation (%)
	Test Suspension (100 mg/L)	Test Suspension (250 mg/L)
0	24.2	12.4
1	10.4	6.9
2	0.0	1.4
3	-6.9	-2.8
4	-10.4	-1.4
5	-10.4	-2.8
6	-13.8	-6.9
7	-17.3	-2.8
8	-13.8	-4.1
9	-20.7	-8.3
10	-20.7	-5.5
11	-20.7	-6.9
12	-17.3	-6.9
13	-24.2	-9.6
14	-24.2	-9.6
15	-24.2	-12.4
16	-20.7	-13.8
17	-24.2	-9.6
18	-27.6	-13.8
19	-27.6	-15.2
20	-24.2	-13.8
21	-27.6	-11.0
22	-27.6	-11.0
23	-24.2	-12.4
24	-27.6	-12.4
25	-27.6	-9.6
26	-31.1	-13.8
27	-31.1	-13.8
28	-27.6	-13.8

Degradation calculated with the mean BOD of the replicates

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Under the conditions of this study, the test material was not biodegradable.

Executive summary:

The biodegradation of the test material was investigated in accordance with the standardised guideline OECD 301F, under GLP conditions.

Activated sludge was exposed to the test material at 100 and 250 mg a.i. test material/L for 28 days, and biochemical oxygen demand was determined.

The average BOD at test day 28 measured in the two test vessels with the test material (low concentration) was 67.9 mg O2/L. The degradation was calculated to be -27.6 % for test suspension at the end of the test. The average BOD at test day 28 measured in the two test vessels with the high concentration of the test material was 65.2 mg O2/L. The degradation was calculated to be -13.8 % for this test suspension at the end of the test.

The BOD measured in the procedure control up to day 28 was 230.8 mg O2/L which corresponds to 91.4 % degradation of the reference material. After 2 days of incubation, the degradation already reached a level of 64.4 % degradation. This rapid degradation of sodium benzoate confirmed the presence of an active microbial population and system integrity. The BOD in the toxicity control (low concentration of test material) was compared with the procedure control. The BOD in the toxicity control was determined to be 154.8 mg 02/L up to day 14 and 187.4 mg 02/L at day 28, which corresponds to 49.8 and 65.3 % degradation, respectively. The BOD in the toxicity control with the high concentration of test material was compared with the procedure control. The BOD in the toxicity control was determined to be 171.1 mg 02/L up to day 14 and 190.1 mg 02/L at day 28, which corresponds to 59.5 and 66.9 % degradation, respectively. Since more than 25 % degradation was observed, the test material was not inhibitory to the inoculum, at both concentrations.

Under the conditions of this study, the test material was not biodegradable.

Description of key information

Under the conditions of this study, the test material was not biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The biodegradation of the test material was investigated in accordance with the standardised guideline OECD 301F, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Activated sludge was exposed to the test material at 100 and 250 mg a.i. test material/L for 28 days, and biochemical oxygen demand was determined.

The average BOD at test day 28 measured in the two test vessels with the test material (low concentration) was 67.9 mg O2/L. The degradation was calculated to be -27.6 % for test suspension at the end of the test. The average BOD at test day 28 measured in the two test vessels with the high concentration of the test material was 65.2 mg O2/L. The degradation was calculated to be -13.8 % for this test suspension at the end of the test.

The BOD measured in the procedure control up to day 28 was 230.8 mg O2/L which corresponds to 91.4 % degradation of the reference material. After 2 days of incubation, the degradation already reached a level of 64.4 % degradation. This rapid degradation of sodium benzoate confirmed the presence of an active microbial population and system integrity. The BOD in the toxicity control (low concentration of test material) was compared with the procedure control. The BOD in the toxicity control was determined to be 154.8 mg 02/L up to day 14 and 187.4 mg 02/L at day 28, which corresponds to 49.8 and 65.3 % degradation, respectively. The BOD in the toxicity control with the high concentration of test material was compared with the procedure control. The BOD in the toxicity control was determined to be 171.1 mg 02/L up to day 14 and 190.1 mg 02/L at day 28, which corresponds to 59.5 and 66.9 % degradation, respectively. Since more than 25 % degradation was observed, the test material was not inhibitory to the inoculum, at both concentrations.

Under the conditions of this study, the test material was not biodegradable.