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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2006-09-25 to 2007-05-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: ISO 14593:2005 " Water Quality - Evaluation of the ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium - Method by Analysis of Inorganic Carbon in Sealed Vessels (CO2-Headspace Test)"
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ISO 10634 as of Nov. 1995: " Water Quality - Guidance for the Preparation and Treatment of Poorly Water-soluble Organic Compounds for the Subsequent Evaluation of their Biodegradability in an Aqueous Medium"
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Analytical purity: 99.18 %;
Lot/batch No.: 23.05.2005;
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge from a biologic sewage treatment plant was used. The chosen plant mostly treats household sewage.
The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf.
Duration of test (contact time):
28 d
Initial conc.:
31.4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Experimental Parameters
The test was conducted with a volume of 150 mL in each test vessel, volume-head space ratio was 3:2. Measurements were made at nine different time points.
For each test series, three replicates for each sampling date were prepared.

150 mL medium were given into the flasks which had been prepared before (BWLE, Test, Toxicity, AbioticLE, RefLE). For the other flasks, the respective solutions were mixed and dosed into the flask to give a total volume of 150 mL. Inoculum was added to all flasks (except for abiotic controls) to give a concentration of suspended solids of 4 ± 1 mg/L. The vessels were closed with silicone septa and lids and were put on the orbital shaker (ca. 120 rpm).
The test was performed at room temperature (22 +/- 2 °C) without direct lighting.
Duration of the test was 28 days. During this time, the created CO2 in the vessels was determined on days 0, 2, 5, 8, 13, 16, 20, 23, 28.
Sampling dates were derived from the degradation behaviour of the test item.
At each sampling date, a blank containing medium only was treated in the same way as the test vessels in order to take into account the IC of the NaOH.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Not performed.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
< 10
Sampling time:
28 d
Details on results:
The attempt was made to determine lag phase (phase from inoculation to 10% degradation), degradation phase (with 10-day-window after reaching 10% degradation) and highest degree of degradation during the test from the graph.
The results for the test item are presented in the following table:
Results
Parameter/Value/Unit
Lag phase: > 28 days
Degradation phase: not detected
10-day-window: not detected
Degradation at the end of 10-day-window: not applicable
Degradation at the end of the test: < 10 %
Results with reference substance:
Day/Reference 1/2/3/Mean
0/-1%/4%/1%/2%
2/68%/66%/68%/67%
5/81%/79%/79%/80%
8/85%/85%/87%/85%
13/93%/92%/92%/92%
16/90%/89%/92%/90%
20/90%/89%/91%/90%
23/96%/91%/88%/92%
28/82%/87%/89%/86%
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item showed no degradation in the CO2 headspace test under current test conditions. All values were in the range of 3 % to 7% degradation. The test item HAT-ISO is therefore considered as “not readily biodegradable”. The result of the test can be considered as valid.
Executive summary:

The test item was tested using a concentration of 31.4 mg HAT-ISO/L in test medium following ISO 14593:2005 in combination with ISO 10634.

Sodium benzoate was used as reference item. Activated sludge from a sewage treating plant was used as inoculum (concentration 4 mg suspended solids/L). The test was left running for 28 days.

All validity criteria were met. Degradation of the reference item was 67 % after two days, the mean inorganic carbon content of the controls at the end of the test was approx. 1 mg/L.

 

The following data were determined for the test item HAT-ISO:

10-day-window:  not detectable

degradation at the end of 10-day-window: not applicable

degradation at the end of the test: < 10 %

 

Therefore, HAT-ISO was not readily biodegradable following ISO 14593:2005.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 2015-09-30 to 2015-12-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study according to internationally accepted guidelines (OECD 310, 2014). A mixture of methanol and isopropanol (ratio 3:1) was used as solvent. Likewise a surface-active agent (Tween 85) was used to achieve a maximum bioavailability of the poorly soluble test item.
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
2014
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
LAUS GmbH, Auf der Schafweide 20, D-67489, Kirrweiler, Germany
Specific details on test material used for the study:
Batch no.: 14299-AKM
Analytical purity: > 99.3 % (HPLC/PDA)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: The active sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf, Germany.
- Aeration: Yes. After the filtration of the sludge, the solids were then resuspended in test medium and aerated.
- Preparation of inoculum for exposure: The inoculum was taken from its source, washed, aerated and the dry matter was determined.
- Filtration: Yes. The sludge was filtrated through a cloth, washed with tap water twice and with test medium once.
- Concentration of sludge: 4 mg/L
Duration of test (contact time):
28 d
Initial conc.:
28.7 mg/L
Based on:
test mat.
Initial conc.:
20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The used mineral medium complies with the test guideline OECD 310.
- Solubilising agent: A stock solution containing 1 g/L test item in a mixture of methanol and isopropanol (ratio 3:1) was prepared. In each test flask 4.3 mL of this solution was added. Then the solvent was completely evaporated using compressed air.
- Other active agents: After addition of the test medium and inoculum, in the test replicates 1 mL of a stock solution containing 304.8 mg/L Tween 85 surface-active agent was added.
- Other Reagents: NaOH, 7-molar (Solution with 280 g NaOH/L)
- Reference items for Carbon Determination: Potassium hydrogenphthalate for total carbon (TC) (p.A., 99.97 %), Na2CO3 (p.A., 99.91 ± 0.05 %) and NaHCO3 (p.A., 99.8 %) for inorganic carbon (IC).
- Test temperature: 20 – 22.8 °C
- pH: 7.4 ± 0.2
- pH adjusted: yes
- Aeration of dilution water: yes
- Suspended solids concentration: The dry matter was determined with 3940 mg suspended solids/L.
- Lighting: The test was performed without direct lighting.

TEST SYSTEM
- Culturing apparatus: Measuring cylinders of 3000 mL nominal volume.
- Culture method: In the measuring cylinders of 3000 mL nominal volume, 2997 mL test medium was mixed with 3.05 mL inoculum for preparation of the control, test item and positive control flask. For preparation of the toxicity control flasks, 1498 mL test medium was mixed with 1.52 mL inoculum. Inoculum was added to all flasks. At last, the vessels were closed with silicone septa and lids and were put on the orbital shaker.
- Pre-treatment: A stock solution containing 1 g/L test item in a mixture of methanol and isopropanol (ratio 3:1) was prepared. In each test flask 4.3 mL of this solution was added. Then the solvent was completely evaporated using compressed air. After addition of the test medium and inoculum, in the test replicates 1 mL of a stock solution containing 304.8 mg/L Tween 85 surface-active agent was added.
- Number of culture flasks/concentration:
Blank control: 2 replicates
Positive Control: 2 replicates
Solvelnt Control: 2 replicates
Toxicity control: 1 replicate
Inorganic Carbon Control: 1 replicate
- Method used to create aerobic conditions: The inoculum was put on the opened orbital shaker.
- Test Vessels: 250 mL-narrow-neck-flasks were used as test vessels. These were closed with lids and silicon septa. In the lid is a small hole, through which the NaOH 7 M was dosed using a needle. All glassware was cleaned with the laboratory cleaning agent and then rinsed with tap water (thrice), diluted HCL (once), tap water (thrice) and diluted water (thrice).
- Test performed in closed vessels: Yes. The vessels were closed with silicone septa and lids and were put on the orbital shaker.
- Test performed in open system: No
- CO2 Determination: Analyses of the emitted CO2 were made by inorganic carbon (IC) measurement. To each vessel to be measured, 1.5 mL of NaOH 7 M was added by syringe and needle and the vessel was shaken for 1 hour again. After settling down, a sample of 1.5 mL was pipetted into TOC-vials which were closed. They were measured two or three times, depending on the variation between the measured values. The carbon analyser was calibrated with freshly prepared reference solutions (containing a mixture of Na2CO3 and NaHCO3) once a week. After every start, quality control samples were measured. At each sampling date, a blank containing mineral medium and NaOH 7 M was measured and the IC of the blank was subtracted from the IC of all sampled vessels.
- Test substance concentration: From the molecular formula, the carbon content was calculated as 69.67 %. The amount to be added to the test flasks was calculated as 4.3 mg test item per 150 mL test medium corresponding to a carbon content of 20 mg/L (corresponding to a test item concentration of 28.7 mg/L).

MEASURING EQUIPMENT
The following instruments and devices were used in the performance of the study.
- Data logger for temperature, ebro
- Analytical scales Mettler Toledo XS DU 205
- Precision scales Mettler Toledo XS 6001S
- Adjustable pipettes with one-way tips Rainin®
- Automatic repeater pipettes with one-way tips, Rainin, Mettler Toledo
- Carbon analyser TOC multi N/C 2100S, Analytik Jena
- pH-meter 3310 wtw
- Heating chamber Heraeus
- Orbital shaker GFL 3019
- Ultra Sonic Bath Sonorex RK 510 H
- Magnetic stirrer
- Fridge
- Glass measuring flasks
- Syringe with needle

SAMPLING
- Sampling method: The medium was prepared from the stock solutions. The inoculum was taken from its source, washed, aerated and the dry matter was determined. The stock solution of test item and positive control were prepared. The test was conducted with a volume of 150 mL in each test vessel. 6 points of measurement were made.
- Sample storage before analysis: The test item was stored in a tightly closed vessel at room temperature (19.5 – 22.8 °C) under dry conditions in the dark.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Solvent control: Yes.
- Toxicity control: Yes. The toxicity control was performed including surface active agent and solvents.
- Positive control: Yes. 1-Octanol (C8H18O), CAS-No. 111-87-5 was used as positive control. From the molecular formula, the carbon content was calculated as 73.8 %. The amount to be added to the test flasks was calculated as 4.1 mg positive control per 150 mL test medium corresponding to a positive control concentration of 27.3 mg/L corresponding to a carbon content of 20 mg/L. Based on a density of 0.83 mg/μL, a volume of 4.9 μL was added directly into the positive control and toxicity control flasks.
- Other: Additionally, one blank control was analysed in order to determine IC coming from medium and/or NaOH.
In the controls the same amount of solvent as in the test replicates was added and also completely evaporated. After addition of the test medium and inoculum, 1 mL of a stock solution containing 300 mg/L Tween 85 (± 10 %) surface-active agent was added. At the end of the test (day 28), six vessels (test series control, positive control, test) resp. three vessels (test series blank, toxicity control) were measured.
Reference substance:
other: 1-octanol
Preliminary study:
The biodegradation of the test item was evaluated in two preliminary studies. The first study was performed according to OECD 301 B “Modified Sturm Test” and the second study was performed according to ISO 14593:2005. In both cases the biodegradation of the test substance was determined to be < 10 %.
Test performance:
This study was performed in order to evaluate the aerobic degradation potential of the test substance by aerobic micro-organisms at a given concentration.
The test item as the sole source of carbon and energy was incubated in a buffer-mineral salts medium which was inoculated with a mixed population of micro-organisms. The test was performed in sealed bottles with a headspace of air, which provides a reservoir of oxygen for aerobic biodegradation. The CO2 evolution resulting from the ultimate aerobic biodegradation of the test item was determined by measuring the IC produced in the test bottles in excess of that produced in blank control vessels containing inoculated medium only. The extent of biodegradation was expressed as a percentage of the theoretical maximum IC production (ThIC), based on the quantity of test item (as organic carbon) added initially.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0.4
Sampling time:
28 d
Details on results:
The degradation of the test item was reached 0.4 % after 28 days, therefore it is considered as not readily biodegradable according to OECD criteria. In addition the test item is regarded as not toxic towards the inoculum in a concentration of 28.7 mg/L as the degradation in the toxicity flask was 94 % at the end of the test.
Results with reference substance:
The reference substance (1-octanol) was biodegraded 81.6 % after 8 days. The validity criteria was met.
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The ready biodegradability of the test substance was examined according to the OECD 310 (CO2-Headspace Test). In this test a mixed population of microorganisms was used as inoculum and in addition a solvent and a surface-active agent were used to achieve a maximum bioavailability of the poorly soluble test item. The test substance was degraded by 0.4 % (mean value) after 28 days. The test substance was found to be not readily-biodegradable under the test conditions.
Executive summary:

The ready biodegradability of the test substance was examined in a test (LAUS, 2015) according to the OECD 310 (CO2-Headspace Test). The biodegradation of the test substance was followed by exposing it to the active sludge from a sewage treating plant in NW-Lachen-Speyerdorf, Germany (concentration: 4 mg suspended solids/L). The chosen plant is treating mostly household sewage. The sludge was filtrated through a cloth, washed with tap water twice and with test medium once. The solids were then resuspended in test medium and aerated. The dry matter was determined with 3940 mg suspended solids/L. The test item was tested using a concentration of 28.7 mg/L, corresponding 20 mg/L organic carbon in test medium. The test item was added in a mixture of methanol and isopropanol (ratio 3:1). After addition the solvent was completely evaporated using compressed air. In addition 1 mL of a stock solution containing 305 mg/L Tween 85 surface-active agent was added. The test was conducted with a volume of 150 mL in each test vessel. 6 points of measurement were made. Two blank controls, two flasks containing 1-Octanol as positive control, two flasks containing test item and one flask containing test item and 1-Octanol (testing for toxic effects) were tested at each point of measurement. Additionally, one blank control was analysed in order to determine the inorganic carbon (IC) coming from medium and/or NaOH. The test was performed at room temperature (20 – 22.8 °C) without direct lighting. The duration of the test was 28 days. During this time, the created CO2 in the vessels was determined on days 0, 8, 15, 21, 26 and 28. Sampling points were derived from the degradation behaviour of the test item.

The degradation of the positive control was determined to be 81.6 % after 8 days. No inhibitory effect on the microorganisms was observed and the mean IC content of the controls at the end of the test was determined to be 1.49 mg/L, therefore all validity criteria were met. The test substance was degraded by 0.4 % (mean value) after 28 days. The test substance was found to be not readily-biodegradable under the test conditions.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1993-11-24 to 1994-10-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
EC 92/69
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Analytical purity: >= 99.65 %
Lot/batch No.: 93.166;
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic, non-adapted
Details on inoculum:
On arrival at the laboratory, the activated sludge will be aerated for four hours. 500 mL of the mixed liquid will be sampled and homogenized for two minutes. It will then settle for 30 minutes.
The supernatant will be decanted to provide sufficient volume for a 1 % inoculum for each CO2 test flask. Viable counts on the supernatant fraction will be performed to determine microbial numbers. This inoculum should normally contain 10E+6 to 20x10E+6 colony forming units per mililitre.
It will be used on the day it is prepared.
Duration of test (contact time):
28 d
Initial conc.:
64.6 other: mg of test article/3 litres
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Sewage micro-organisms (concentration: 54x10E2 colony forming units/mL - Supplier: Station de traitement des eaux usées, Pierre-Bénite) were exposed to the test article for 28 days, in comparison with control and reference substance.
Three series of flaks were used in parallel:
- Test inoculum: Test article at a concentration of 21.52 mg/L corresponding to 15 mg of T.O.C (Total Organic Carbon) per litre (2 flasks);
- Reference inoculum: Reference substance at 100 mg/litre, corresponding to 17.65 mg TOC per litre (1 flask);
- Blank inoculum: One control assay in the absence of any substance (2 flasks).
Reference substance:
acetic acid, sodium salt
Preliminary study:
Not performed
Test performance:
The 'Modified Sturm Test' uses carbon dioxide production as the primary end point in assessing the biodegradation of organic chemicals. A measured volume of inoculated mineral medium, containing a known concentration of the test substance (10-20 mg DOC or TOC/l) as the nominal sole source of organic carbon is aerated by the passage of carbon dioxide-free air at a controlled rate in the dark or in diffuse light. Degradation is followed over 28 days by determining the carbon dioxide produced. The CO2 is trapped in barium or sodium hydroxide and is measured by titration of the residual hydroxide or as inorganic carbon. The amount of carbon dioxide produced from the test substance (corrected for that derived from the blank inoculum) is expressed as a percentage of ThCO2. The degree of biodegradation may also be calculated from supplemental DOC analysis made at the beginning and end of incubation.
.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
11
Sampling time:
28 d
Details on results:
Points of degradation plot (test substance):
2 % degradation after 3 d
3 % degradation after 7 d
7 % degradation after 14 d
8 % degradation after 19 d
11 % degradation after 28 d
Results with reference substance:
Points of degradation plot (reference substance):
14 % degradation after 3 d
39 % degradation after 7 d
62 % degradation after 14 d
75 % degradation after 19 d
84 % degradation after 28 d

CO2 production and % biodegradation:

 

 

Test article

Reference substance (sodium acetate)

Time (Day)

Cumulative CO2production (mg)

% biodegradation

Cumulative CO2production (mg)

% biodegradation

3

2.75

2

27.50

14

7

5.50

3

75.02

39

14

12.10

7

121.33

62

19

13.09

8

146.63

75

28

17.27

11

162.58

84

 

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the experimental conditions, HAT ISO can be considered as not readily biodegradable.
Executive summary:

HAT ISO was tested for ready biodegradability using the modified Sturm test. The evaluation of the biotic degradation showed 11 % biodegradation after 28 days. HAT ISO can be considered as not readily biodegradable.

Description of key information

The test substance was tested for biotic degradation in the carbon dioxide evolution test according to EU Guideline C.4 and in the aerobic biodegradation test according to the guidelines ISO 14953 and ISO 10634. HAT-ISO showed a biodegradation of 11 % within 28 days in the carbon dioxide evolution test. In the CO2 Headspace test, according to the ISO standards, HAT-ISO showed a biodegradation of < 10 % within 28 days. Because the test substance was not biodegraded in the above mentioned tests, a supplementary test (LAUS, 2015) with other supposed better conditions for the biodegradation (mixed population of microorganisms as inoculum, solvent and surface-active agent used) was conducted according to the OECD 310 (CO2-Headspace Test). In this test the test substance was degraded by 0.4 % (mean value) after 28 days. Based on the available data and the test results, the test substance was determined to be not readily biodegradable under the test conditions.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The test item was tested for ready biodegradability in a test (Pharmacon Europe, 1994) using the modified Sturm test. The biodegradation of the test substance was followed by exposing it to the non-adapted active sludge from a domestic sewage treating plant after 28 days. The test item was tested using a concentration of 21.5 mg/L. The evaluation of the biotic degradation showed 11 % biodegradation after 28 days.

In addition the biodegradation of the test item was evaluated in a test (LAUS, 2007) using a concentration of 31.4 mg HAT-ISO/L in a test medium following ISO 14593:2005 in combination with ISO 10634. Sodium benzoate was used as reference item. Activated sludge from a sewage treating plant was used as inoculum (concentration 4 mg suspended solids/L). The test was left running for 28 days. The degradation of the reference item was determined to be 67 % after two days and the mean inorganic carbon content of the controls at the end of the test was determined to be approx. 1 mg/L. All validity criteria were met. The test substance was degraded by < 10 % (mean value) after 28 days.

 

Because the test substance was not biodegraded in the above mentioned tests, a supplementary test (LAUS, 2015) with other supposed better conditions for the biodegradation was conducted according to the OECD 310 (CO2-Headspace Test). In this test a mixed population of microorganisms was used as inoculum and in addition a solvent (mixture of methanol and isopropanol (ratio 3:1)) and a surface-active agent (Tween 85) were used to achieve a maximum bioavailability of the poorly soluble test item. The biodegradation of the test substance was followed by exposing it to an active sludge from a sewage treating plant in NW-Lachen-Speyerdorf, Germany (concentration: 4 mg suspended solids/L). The concentration of the test substance was 28.7 mg /L, corresponding 20 mg/L organic carbon in the test medium. The test item was added in a solvent (mixture of methanol and isopropanol (ratio 3:1)) and after the addition the solvent was completely evaporated using compressed air. Supplementary 1 mL of a stock solution containing 305 mg/L Tween 85 surface-active agent was added. 1-Octanol was used as positive control. In the control the same amount of solvent as in the test replicates was added and also completely evaporated. The degradation of the positive control was determined to be 81.6 % after 8 days. No inhibitory effect on the microorganisms was observed and the mean inorganic carbon (IC) content of the controls at the end of the test was determined to be 1.49 mg/L, therefore all validity criteria were met. The test substance was degraded by 0.4 % (mean value) after 28 days.

 

Based on the available data and the test results, the test substance was determined to be not readily biodegradable under the test conditions.