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Environmental fate & pathways

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 2017-07-20 to 2017-08-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017
Reference Type:
other: amendment to the study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Municipal sewage treatment plant, 31137 Hildesheim, Germany.
Activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste. (Receipt : 2017-07-13)

- Preparation of inoculum for exposure: Colony forming units (CFU) of the inoculum for the Modified Sturm Test were determined prior to test start by standard dilution plate count: approx. 4.1 x 10^9 CFU/L, corresponding to approx. 4.1 x 10^7 CFU/L in the test vessel. The concentration of the test item and the theoretical CO2 production (ThCO2) were calculated based on the carbon content.

The necessary amounts of ultrapure water, mineral salts medium and inoculum were placed in each incubation vessel. The vessels were aerated for 24 h with CO2 free air.

- Initial cell/biomass concentration: Approx. 10^7 - 10^8 CFU/L
Duration of test (contact time):
ca. 28 d
Initial test substance concentrationopen allclose all
Initial conc.:
ca. 29 mg/L
Based on:
test mat.
Initial conc.:
ca. 10.4 other: mg C/L
Based on:
TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral salts medium according to OECD 301B/CO2 evoluation test.

- Test temperature: measured range between 20.5 and 24°C
- Aeration of dilution water: 30 - 100 mL/min
- Continuous darkness: Low light conditions (brown glass bottles)


TEST SYSTEM
- Culturing apparatus: brown glass vessel (capacity 5000mL), where 3000mL of test medium is added. Continous stirring of the medium during the experiment.
- Number of culture flasks/concentration:
* Functional control (Sodium benzoate) : 1 replicate
* Toxicity control: 1 replicate
* Inoculum control : 2 replicates
* Test item : 2 replicates

- Method used to create aerobic conditions: The vessels were aerated for 24 h with CO2 free air. After 24 h the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution.


CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium without test and/or reference item
- Toxicity control: Test item and reference item in test concentration
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
The study was performed according to OECD 301 B and GLP Guidelines. The validity criteria were fulfilled according to the guideline:
•The total CO2 evolution in the inoculum control at the end of the test was 20.4 mg/L
(validity criterion: < 40 mg CO2 /L after 28 days).
•The degradation of the functional control reached the pass level of >= 60% within 8 days (degradation: 70% on day 8).
•The differences of extremes of replicate values of removal of the test item at the end of the test was less than 20% (11% difference on day 28).
•The degradation of the toxicity control reached the pass level of 25% within 6 days (degradation: 32% on day 6).
% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 21
Sampling time:
28 d
Details on results:
The mean of replicates reached the 10% level (beginning of biodegradation) on day 11. Both test item replicates did not reach the 60% pass level within the 28-day period of the study. The mean biodegradation on day 28 was 21%.

Any other information on results incl. tables

Table 1:   CO2-Production and Biodegradation after 28 Days

CO2 Production Functional Control Test Item Toxicity Control test item +  reference item
1 2
Net[mg/3 L] 121.2 17.4 29.6 122.9
[mg/L] 40.4 5.8 9.9 41.0
Theor.[mg/3 L] 127.8 114.8 242.6
[mg/L] 42.6 38.3 80.9
Degradation [%] 95 15 26 51

Table 2:   CO2-Production and Biodegradation in the Inoculum Control, the Functional
                  Controland the Toxicity Control

Study Day Date Inoculum control Functional Control Toxicity Control
[mg CO2/3 L] [mg CO2/3 L] Degr. [mg CO2/3 L] Degr.
mv gross net  % gross net  %
1 21/07/2017 3.0 13.8 10.8 8 3.9 0.9 0
4 24/07/2017 6.3 40.2 44.7 35 51.9 46.5 19
6 26/07/2017 7.0 35.9 73.6 58 37.1 76.6 32
8 28/07/2017 7.0 23.2 89.8 70 23.9 93.5 39
11 31/07/2017 5.1 17.6 102.3 80 10.9 99.3 41
14 03/08/2017 5.5 14.3 111.1 87 12.3 106.1 44
18 07/08/2017 5.3 11.0 116.8 91 11.2 112.0 46
22 11/08/2017 6.9 9.8 119.7 94 11.8 116.9 48
25 14/08/2017 5.5 5.9 120.1 94 9.9 121.3 50
28 17/08/2017 4.4 5.3 121.0 95 5.5 122.4 50
29* 18/08/2017 5.1 5.3 121.2 95 5.6 122.9 51

Degr. = degradation                              mv = mean value           *) results of last two gas wash bottles

 

In the inoculum controlthe total CO2production was 20.4 mg CO2/L after 28 days.

Table 3: O2-Production and Biodegradation in the Inoculum Control and Test Item Samples

Study Day Date Inoculum Control Test Item
Replicate 1 Replicate 2
[mg CO2/3 L] [mg CO2/3 L] Degr. [mg CO2/3 L] Degr.
mv Gross Net Sum [%] Gross Net Sum [%]
1 21/07/2017 3.0 3.6 0.6 1 4.3 1.3 1
4 24/07/2017 6.3 11.0 5.3 5 13.5 8.5 7
6 26/07/2017 7.0 8.6 6.9 6 10.5 12.0 10
8 28/07/2017 7.0 9.8 9.7 8 7.9 12.9 11
11 31/07/2017 5.1 5.8 10.4 9 6.1 13.9 12
14 03/08/2017 5.5 6.5 11.4 10 7.9 16.3 14
18 07/08/2017 5.3 8.4 14.5 13 9.9 20.9 18
22 11/08/2017 6.9 9.4 17.0 15 11.7 25.7 22
25 14/08/2017 5.5 5.1 17.0 15 6.9 27.1 24
28 17/08/2017 4.4 4.8 17.4 15 5.8 28.5 25
29* 18/08/2017 5.1 3.4 17.4 15 6.2 29.6 26

 

Table 4 : pH-Values on Day 28

Inoculum Control

Func-tional
Control

Test Item

Toxicity Control

No. 1

No. 2

No.1

No. 1

No. 2

No. 1

7.72

7.69

7.77

7.65

7.67

7.76

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the test conditions the test item is classified as not readily biodegradable within the 28 day period of the study.
Executive summary:

The ready biodegradability of the test item was determined with a non-adapted activated sludge over a test period of 28 days in the Modified Sturm Test. The study was conducted from 2017-07-20 to 2017-08-18, according to OECD 301 B at the test facility.

The test item was tested at a concentration of 29 mg/L with 2 replicates corresponding to a carbon content (TOC) of 10.4 mg C/L in the test vessels.The test vessels were incubated at low light conditions and at a temperature of 22 ± 2 °C.

The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO2 produced by the respiration of bacteria. The degradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29 after residual CO2 had been purged from the test solutions over a period of 24 hours. The percentage CO2 production was calculated in relation to the theoretical CO2 production (ThCO2) of the test item. The biodegradation was calculated for each titration time.

 

To check the activity of the test system sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60% within 8 days and a maximum biodegradation of 95% on day 28.

 

In the toxicity control containing both test and reference item a biodegradation of 44% was determined within 14 days and it came to 51% after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

 

The biodegradation of the test item is shown graphically in Figure 1 in comparison to the readily degradable functional control and the toxicity control. The mean of replicates reached the 10% level (beginning of biodegradation) on day 11. Both test item replicates did not reach the 60% pass level within the 28-day-period of the study. The mean biodegradation on day 28 was 21%.

Under the test conditions the test item is classified as not readily biodegradable within the 28 day period of the study.