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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-10-2011 - 10-11-2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Principles of method if other than guideline:
This test has been performed according to slightly modified EEC, OECD and ISO Test Guidelines (OECD, 1992; EU, 1992; ISO, 1994)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name Halamid® PG
Chemical name sodium N-chloro-para-toluenesulfonamide
CAS no 127-65-1
Batch/lot No. 1012692080
Purity 98.9%
Solubility in water soluble
Stability not relevant
Storage at ambient temperature in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
natural water: freshwater
Details on inoculum:
River water was sampled from the Rhine near Heveadorp, The Netherlands (06-
10-2011). The nearest plant (Arnhem-Zuid) treating domestic wastewater
biologically was 3 km upstream. The river water was aerated for 7 days before
use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River
water without particles was used as inoculum. The particles were removed by
sedimentation after 1 day while moderately aerating.
Duration of test (contact time):
28 d
Initial conc.:
1 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with
glass stoppers.

Nutrients, stocks and administration
The river water used in the Closed Bottle test was spiked per liter of water with
8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2 HPO4·2H2O, 22.5 mg
MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was
omitted from the medium to prevent nitrification. Sodium acetate and the test
substance were added to the bottles using stock solutions of 1.0 g/L.

Test procedures
The Closed Bottle test was performed according to the study plan. The study plan
was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing
only inoculum, 10 bottles containing inoculum and test substance, and 6
bottles containing sodium acetate and inoculum. The concentrations of the test
substance and sodium acetate in the bottles were 1.0 and 6.7 mg/L, respectively.
Each of the prepared solutions was dispensed into the respective group of BOD
bottles so that all bottles were completely filled without air bubbles. The zero time
bottles were immediately analyzed for dissolved oxygen using an oxygen
electrode. The remaining bottles were closed and incubated in the dark. Two
duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen
concentration at day 7, 14, 21, and 28.

Analyses
The dissolved oxygen concentrations were determined electrochemically using an
oxygen electrode (WTW TrioXmatic EO 200) and meter (WTW OXI 530) (Retsch,
Ochten, The Netherlands). The pH was measured using a Eutech Cyberscan pH11
pH meter (Eutech Instruments, Nijkerk, The Netherlands). The temperature was
measured and recorded with a sensor connected to a data logger.
Reference substance:
acetic acid, sodium salt
Test performance:
Validity of the test
The validity of the test is demonstrated by an endogenous respiration of 1.4 mg/L at day 28 (Table I). Furthermore, the differences of the replicate values
at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 76. Finally, the validity of the test is
shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Key result
Parameter:
% degradation (O2 consumption)
Value:
92
Sampling time:
28 d
Remarks on result:
other: based on ThODNH3
Key result
Parameter:
% degradation (O2 consumption)
Value:
73
Sampling time:
28 d
Remarks on result:
other: based on ThODNO3
Details on results:
Theoretical oxygen demand (ThOD)
The calculated theoretical oxygen demands (ThOD) of Halamid® PG are 1.2 mg/mg (substance oxidized to carbon dioxide, water and ammonium) and 1.5
mg/mg (substance oxidized to carbon dioxide, water and nitrate). The ThOD of sodium acetate is 0.8 mg/mg.

Toxicity
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because
possible toxicity of Halamid® PG to microorganisms degrading acetate is not relevant. Halamid® PG was added to the bottles at a concentration of 1.0 mg/L to
avoid toxic effects of this substance to the competent bacteria (bacteria capable of degrading Halamid® PG. Inhibition is usually detected prior to the onset of the biodegradation through suppression of the oxygen consumption in the presence of a test substance. Inhibition of the endogenous respiration of the inoculum by the test substance in the presence of Halamid® PG was detected at day 7 although the initial test substance concentration was 1.0 mg/L. Inhibition of the biodegradation in the test due to the "high" initial concentration of the test substance is therefore expected. Nonetheless, degradation of Halamid® PG was detected at day 14. Under environmental conditions, Halamid® PG is unlikely to pose a toxicity risk to the competent microorganisms because this substance will be present in the environment in the ug/L range.

Test conditions
The pH of the media was 8.1 at the start of the test. The pH of the medium at day 28 did remain 8.1 (control and test). Temperatures were within the prescribed temperature range of 22 to 24°C.

Biodegradation
Halamid® PG is biodegraded by 92%(ThODNH3) and 73 (ThODNO3) at day 28 in the Closed Bottle test (Figure and Tables I-II). Halamid® PG is a substance with one nitrogen atom. Nitrogen-containing organic substances are biodegraded by competent microorganisms to carbon dioxide, water, ammonium. The
biodegradation percentages calculated with ThODNH3 therefore already represents the complete degradation of Halamid® PG. The ammonium formed is
subsequently oxidized to nitrate by nitrifying bacteria. Nitrifying bacteria are not involved in the biodegradation of Halamid® PG. The biodegradation percentage
calculated with the ThODNO3 represents the degradation of Halamid® PG and the subsequent oxidation of ammonium. The time window was therefore determined using the biodegradation curve calculated with the ThODNH3. The time window of approximately 12 days obtained with ThODNH3 does reflect the biodegradation rate of Halamid® PG (Figure). The time window criterion for the Closed bottle test (<14 days) was therefore met. Halamid® PG should therefore be classified as readily biodegradable.

Mean (n=2) dissolved oxygen concentrations (mg/L) in the closed bottles

Time (days)

Oxygen concentration (mg/L)

 

Ot

Oc

Oa

0

8.9

8.9

8.9

7

8.5

8.2

4.5

14

7.7

7.9

3.8

21

7.1

7.7

 

28

6.4

7.5

 

Oc Mineral nutrient solution with only inoculum.

Ot Mineral nutrient solution with test material (1.0 mg/L), and inoculum.

Oa Mineral nutrient solution with sodium acetate (6.7 mg/L) and with inoculum.

Oxygen consumption (mg/L) and the percentages biodegradation of the test substance, Halamid PG (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle test.

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

Test substance

Acetate

Test substance

NH3 - NO3

Acetate

0

0.0

0.0

0 - 0

0

7

-0.3 (toxic)

3.7

0 - 0

69

14

0.2

4.1

17 - 13

76

21

0.6

 

50 - 40

 

28

1.1

 

92 - 73

 

Validity criteria fulfilled:
yes
Remarks:
see test performance for details
Interpretation of results:
readily biodegradable
Conclusions:
Tosylchloramide sodium was biodegraded by 73% (ThODNO3) or 92% (ThODNH3) at day 28 in the Closed Bottle test and met the time window, so should be classified as readily biodegradable.
Executive summary:
The ready biodegradability was determined in the Closed Bottle test (OECD TG 301D) performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with GLP. Inhibition of the endogenous respiration of the inoculum by the test substance was detected at day 7 although the initial test substance concentration was 1.0 mg/L. Inhibition of the biodegradation in the test due to the "high" initial concentration of the test substance is therefore expected. Nonetheless, degradation of Tosylchloramide sodium was detected at day 14. Under environmental conditions,

Tosylchloramide sodium is unlikely to pose a toxicity risk to the competent microorganisms because this substance will be present in the environment in the ug/L range. The substance was biodegraded by 73% (ThODNO3) or 92% (ThODNH3) at day 28 and met the time window, so should be classified as readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
nov-2009 Jan-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The source of test organisms was secondary effluent freshly obtained from a municipal sewage treatment
plant: 'Waterschap de Maaskant', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Secondary effluent was filtered through a coarse filter paper, the first 200 ml was discarded. The filtrate was
kept aerated until inoculation.

Inoculation: 4 ml filtrate of secondary effluent per litre of final volume.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Initial conc.:
4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test duration: 28 days for the inoculum blank and test suspensio, 14 days for the procedure and toxicity control
Test bottles 250-300 ml BOD bottles with glass stopper
Test set up:
- lnoculum blank Containing only inoculum (no test substance
- Procedure control Containing reference substance and inoculum
- Test suspension Containing test substance and inoculum (applicable for both test concentrations)
- Toxicity control Containing test substance at the lowest concentration, reference substance and inoculum

Number and groups of test bottles
Individual BOD bottles were prepared for each measuring point, except that the bottle measured at the start was also measured on day 7.
Parallel groups were prepared to allow duplicate measurements of oxygen consumption at the test intervals.

Illumination: The test bottles were protected from light.

p-toluenesulfonamide (PTSA) was a white crystalline solid with a purity of 99.9%. Nominal concentrations of 2 and 4 mgll were tested.
Preparation of the test media started with a stock solution of 100 mgll by adding 99.7 mg test substance to 1000 ml of mineral medium.
Combined ultra sonic treatment and thorough mixing (30 min) was used to accelerate dissolution of the test substance in mineral medium.
Exact volumes of the clear and colourless stock solution corresponding to the test concentrations and microbial organisms were added to the test
medium.

Determination of oxygen concentration:
Frequency: In duplicate; immediately at the start of the experiment (day O), and at day 7, 14, 21 and 28.

pH measurement: at the start of the test
Temperature measurement: continously

Reference substance:
acetic acid, sodium salt
Remarks:
>99% purity; batch no K34333668
Parameter:
% degradation (O2 consumption)
Value:
86
Sampling time:
28 d
Remarks on result:
other: 2 mg/l test substance
Parameter:
% degradation (O2 consumption)
Value:
45
Sampling time:
28 d
Remarks on result:
other: 4 mg/l test substance
Details on results:
The ThOD of p-toluenesulfonamide (PTSA) was calculated to be 1.68 mg O2 per mg.
The ThOD of sodium acetate (reference substance) was calculated to be 0.78 mg 02/mg.

Mean biodegradation percentages at different point in time

Test medium

Conc (mg/l)

% biodegradation after x days

 

7

14

21

28

control

 

73

70

 

 

Test substance low

2

0

9

66

86

Test substance high

4

-1

-1

25

45

Toxicity control

 

21

31

 

 

The relative biodegradation values calculated from the O2 measurements performed during the test period of 28 days revealed 86 and 45% biodegradation (mean values) of p-toluenesulfonamide (PTSA) for the lowest (2 mg/l) and highest (4 mg/l) concentration respectively. Furthermore, biodegradation of at least 60% was reached within 14 days of biodegradation exceeding 10%. In the toxicity control more than 25% biodegradation occurred within 14 days (31% based on ThOD). Thus, the toxicity control showed that p-toluenesulfonamide (PTSA) did not inhibit microbial activity at 2 mg/l. Since the biodegradation in the highest concentration (4 mgll) was not completely in line with the biodegradation in the lowest concentration (2 mgll), there might have been a slight toxic effect at the highest concentration. This effect has no effect on the outcome of the study.

Temperature of test room varied between 21.8 and 22.3 'C

pH values at start of test varied between 7.3 -7.4

Acceptability of the test

1. Oxygen depletion in the inoculum blank was below 1.5 mg 02/1 after 28 days (0.93 mg 02/1, see Table 2)

2. The residual concentration of oxygen in the test bottles was > 0.5 mg/l at any time (see Table 2).

3. The differences of duplicate biodegradation values were less than 20% at the end of the test and at the end of the 14-day window

4. The control substance was biodegraded by at least 60% (mean value: 70%) within 14 days (see Table 1).

Since allcriteria for acceptability of the test were met, this study was considered to be valid.

Validity criteria fulfilled:
yes
Remarks:
see overall remarks
Interpretation of results:
readily biodegradable
Conclusions:
Study performed under GLP, according guidelines, meeting quality and validity criteria.
p-toluenesulfonamide (PTSA) biodegraded 86% in the closed bottle test at day 28, meeting als the time- window criteria, and should therefore be clasified as readily biodegradable.
Executive summary:

Ready Biodegradability: closed bottle test with p-toluenesulfonamide (PTSA).

The study procedure described in this report were based on the OECD guideline No. 301 D, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. No. 44012008 of 30 May 2008, Publication No. L220, Part C.4-E and the IS0 International Standard 10707, 1994.

p-toluenesulfonamide (PTSA) was a white crystalline solid with a purity of 99.9%. Nominal concentrations of 2 and 4 mg/l were tested. Theoretical oxygen demand of p-toluenesulfonamide (PTSA) was calculated to be 1.68 mg 02/mg. The following test setup was applied:

- lnoculum blank (only inoculum, no test substance, duplicate measurements);

- Procedure control (sodium acetate and inoculum, duplicate measurements);

- Test suspension (p-toluenesulfonamide (PTSA) and inoculum, duplicate measurements);

- Toxicity control (sodium acetate, p-toluenesulfonamide (PTSA) and inoculum, duplicate measurements).

Preparation of the test media started with a stock solution of 100 mgll by adding 99.7 mg test substance to 1000 ml of mineral medium. Combined ultra sonic treatment and thorough mixing (30 min) was used to accelerate dissolution of the test substance in mineral medium. Exact volumes of the clear and colourless stock solution corresponding to the test concentrations and microbial organisms were added to the test medium. Measurements was performed at the start of the experiment (day 0) and at day 7, 14, 21 and 28. The relative biodegradation values calculated from the O2 measurements performed during the test period of 28 days revealed 86 and 45% biodegradation (mean values) of p-toluenesulfonamide (PTSA) for the lowest (2 mg/l) and highest (4 mg/l) concentration respectively. Furthermore, biodegradation of at least 60% was reached within 14 days of biodegradation exceeding 10%. In the toxicity control more than 25% biodegradation occurred within 14 days (31%). Thus, the toxicity control showed that p-toluenesulfonamide (PTSA) did not inhibit microbial activity at 2 mg/l. Since the biodegradation in the highest concentration (4 mg/l) was not completely in line with the biodegradation in the lowest concentration (2 mg/l), there might have been a slight toxic effect at the highest concentration. This effect has no effect on the outcome of the study. Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, p-toluenesulfonamide (PTSA) is designated as readily biodegradable.

Description of key information

Both for Tosylchloramide sodium as for the first primary degradate p-TSA readily biodegradability is concluded.

For Tosylchloramide sodium and for p-TSA guideline readily biodeg studies were performed both showing ready biodegradability, (73 -92% and 86% biodeg in 28d respectively).

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information