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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:
20 Jun - 12 Jul 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
1998
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss Federal Office of Public Health, Bern, Switzerland

Test material

Reference
Name:
Unnamed
Type:
Constituent

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: local wastewater treatment plant, ARA Birs, Birsfelden, Switzerland (predominantly domestic sewage)
- Storage conditions: Based on the measured dry weight of suspended solids, the wet sludge was suspended in mineral medium and adjusted to a concentration equivalent to 4 g dry material per liter. During the holding period the sludge was aerated at room temperature.
- Storage length: 1 d prior use
- Preparation of inoculum for exposure: Prior to use, the dry weight of the suspended solids was determined again and readjusted to 4 g dry material per liter. Thereafter the sludge was diluted with mineral medium to a concentration of 1 g dry material per liter. Defined volumes of this diluted activated sludge were added to mineral medium to obtain a final concentration of 30 mg dry material per liter.
- Pretreatment: The aerobic activated sewage sludge was washed twice with tap water by centrifugation and decantation of the supernatant liquid phase. Finally the solid material was resuspended in mineral medium. Aliquots of the homogenized final sludge suspension was weighed, thereafter dried and dry weight the suspended solids was determined.
Duration of test (contact time):
19 d
Initial test substance concentrationopen allclose all
Initial conc.:
15 mg/L
Based on:
TOC
Initial conc.:
20.8 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
See also section below "Any other information on materials and methods incl. tables"

TEST CONDITIONS
- Composition of medium: mineral medium, according to the guideline; details in "Any other information on materials and methods incl. tables"
- Additional substrate: no
- Test temperature: 22 °C
- pH: 7.4 (start); 7.3 - 7.4 (end); see Table 4
- pH adjusted: no
- Aeration of dilution water: CO2-free air
- Suspended solids concentration: 30 mg/L (final concentration)
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 L amber glass bottles, each fitted with an aeration glass tube reaching nearly the bottom of the vessel and an outlet were used as test vessels.
- Number of culture flasks/concentration: 2 for Test item; 2 for Procedure control, 2 for Inoculum control an 1 for Toxicity control
- Method used to create aerobic conditions: Compressed CO2-free air was bubbled through the solution in each test vessel at a rate of 30-100 mL/min. The test media were aerated overnight.
- Measuring equipment: CO2 absorption flasks and TOC infrared gas analyzer equipped with an automatic sampler (i.e. vario TOC cube from Elementar Analysensysteme GmbH, Hanau, Germany)
- Details of trap for CO2: The CO2 evolved from the test vessels were collected in 2 x 500 mL Drechsel bottles (gas-absorbtion flasks) containing 300 and 200 mL of 0.05 M NaOH. The air leeding connections between the test vessel and the absorption flasks were done by glass tubes.

SAMPLING
- Sampling frequency: For analysis of the evolved CO2, samples were taken on exposure day 2, 5, 7, 9, 12, 14, 16, 19 (before acidification), and 20 (after driving off residual CO2). For the sampling on exposure day 0, instead of sampling the first and second absorber flask for each test vessel, the inorganic carbon (IC) content of the 0.05 M NaOH solution used to fill all the absorber flasks was measured.
- Sampling method: On each sampling day, an aliquot of 10.0 mL was withdrawn from the absorber flask nearest to the test vessel for analysis of inorganic carbon (IC). Additional samples for analysis of IC were withdrawn from the second absorber flask of all test vessels on exposure day 14 and at the end of the exposure period on exposure day 19 in order to correct for any carry over of CO2. After sampling on exposure day 19, the pH was measured in each test vessel. Next, 1 mL of concentrated HCl was added to each test vessel and the vessels were aerated overnight with CO2-free air to drive off any residual CO2 present. On day 20, a sample from each absorber flask was withdrawn and analyzed for IC to determine residual CO2 that was present in the test suspensions on exposure day 19. In this way, any residual CO2 remaining in the test suspensions was determined as the difference between the amounts of IC found before and after acidification.
- Other: At the start of the test (day 0), inorganic and total carbon (IC and TC, respectively) were measured in the test and inoculum vessels.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 replicates
- Toxicity control: yes, 1 replicate
- Procedure control (reference substance): yes, 2 replicates
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
Prior to test start, the solubility of the test item in mineral medium was checked, and the test item was found to be not soluble at a concentration of 103.1 mg/L. Therefore, a test item stock solution could not be prepared and the test item was added directly to the test vessels.
% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
85
Sampling time:
19 d
Remarks on result:
other: Readily biodegradable
Remarks:
In accordance with the test guidelines the test was ended after 19 days since the biodegradation curves of the test item had reached a plateau over at least three determinations.
Details on results:
The calculation of the percent biodegradation of the test item was based on a calculated total organic carbon content (TOC) of 0.720 mg C/mg test item.
The CO2 formation of the test substance in the test media significantly increased from the start of the test until exposure day 14, when a plateau was reached. At the end of the 19 day exposure period, average biodegradation was 85 %.
The pass level for ready biodegradability, i.e. a CO2 formation of at least 60 % of the TOC in a 10-day window within the normally 28-day period of the test was reached by Exposure Day 7 (62 %).

In conclusion, the test item was found to be readily biodegradable under the test conditions within 19 days.

BOD5 / COD results

Results with reference substance:
The evaluation of the percent biodegradation of the reference item sodium benzoate was based on a calculated total organic carbon content (TOC) of 0.583 mg C/mg sodium benzoate.
In the procedure controls, average biodegradation of the reference item was 72 % by exposure day 5, and 90 % by exposure day 14, thus confirming suitability of the activated sludge (≥ 60 % degradation by exposure day 14). By the end of the test (exposure day 19), average biodegradation was 92 %.

Any other information on results incl. tables

Toxicity Control

The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the sum of the total organic carbon content (TOC) of the test item and the reference item.

In the toxicity control, the CO2 formation over the 19 day exposure period correlated very well with the CO2 evolution from the two added substances, i.e. test item and reference item. Within 14 days of exposure, biodegradation amounted to 78 %, reaching 83 % at the end of the test. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 20.8 mg/L because biodegradation in the toxicity control was ≥ 25 % within 14 days of incubation.

 

TABLES

The tabulated values represent rounded results obtained by calculation using the exact raw data.

Table 1     Inorganic Carbon (IC Concentrations) Measured in the Absorber Flasks

IC found in absorber flasks (ICabs, mg C/L)  
Test item Reference item   Toxicity control Inoculum control  
Time (days) / absorber flask no. Replicate No. 1 Replicate No. 2 Replicate No. 1 Replicate No. 2 Replicate No. 1 Replicate No. 1 Replicate No. 2
2/1 40.9 38.9 92 90.4 114.5 12.6 12.1
5/1 104.5 95.3 126.9 126.4 179.4 22.3 23.1
7/1 121.6 110.3 136.9 136.6 195.9 25.9 27.1
9/1 135.8 125.7 148.3 146.1 213.8 28.6 32.4
12/1 153 140.8 158.6 157.6 237 35.6 36.1
14/1 158.6 152 164.4 161.8 246 37.2 38.3
14/2 18.3 29.9 27.8 25.5 56.2 6.9 8.1
16/1 162.5 154.9 166.6 160.7 252.7 41.1 42.4
19/1 177.6 158.5 175.1 172.4 266.6 44.6 46.8
19/2 20.1 32.7 30.1 26.5 67.2 7.9 8
20/1 174.6 166 175.3 173 265.7 45.6 48.4
20/2 20.7 34.3 29 26.7 66.4 6.9 7.8

Table 2     Absolute Amount of Inorganic Carbon (IC) Produced

IC formation per test vessel (ICprod, mg C/3 L)
Test item   Reference item   Toxicity control Inoculum control  
Time (days) Replicate No. 1 Replicate No. 2 Replicate No. 1 Replicate No. 2 Replicate No. 1 Replicate No. 1 Replicate No. 2
2 12.8 12.5 28.4 27.8 36 4 3.9
5 32 30.2 39.7 39.4 57.2 7.1 7.4
7 37.3 35.2 43.3 43 63.4 8.3 8.8
9 41.7 40.2 47.2 46.3 69.8 9.2 10.4
12 46.9 45.4 51 50.3 78.3 11.3 11.7
14 48.9 49.1 53.3 52.1 82.1 11.9 12.5
16 49.9 50 54 51.9 84.6 12.9 13.4
19 53.6 51.1 56.2 54.7 89 13.8 14.4
20 53.1 53.1 56.1 54.9 88.7 13.8 14.7

Table 3     Biodegradation of the Test Item and the Reference Item

% Degradation1
Test item   Reference item    Toxicity control
Time (days) Replicate No. 1 Replicate No. 2 Mean Replicate No. 1 Replicate No. 2 Mean Replicate No. 1
2 19.7 19.1 19.4 54.4 53.2 53.8 35.6
5 55.1 50.9 53 72.2 71.4 71.8 55.5
7 64 59.3 61.7 77.3 76.6 76.9 61
9 70.9 67.6 69.2 83 81 82 66.7
12 78.7 75.3 77 87.8 86.3 87 74.2
14 81.5 81.9 81.7 91.3 88.7 90 77.7
16 81.7 81.8 81.8 90.7 86.1 88.4 79.4
19 87.9 82.3 85.1 93.6 90.3 92 83.3

1:            Based on values presented in Table 2, corrected for the inoculum controls

Table 4     pH of the Test Solutions or Suspensions

Replicate No. Identification pH Start pH End
1 Test item 7.4 7.3
2 Test item 7.4 7.3
1 Procedure control 7.4 7.4
2 Procedure control 7.4 7.4
1 Toxicity control 7.4 7.3
1 Inoculum control 7.4 7.3
2 Inoculum control 7.4 7.3

 

Figure 1:    Biodegradation of the Test Item 10-HSA and the Reference Item Sodium benzoate during the Incubation Period

A: Overview over the whole test series

B: 10-day window for the biodegradation of the test item

(see picture below under section Illustration)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
In a valid, reliable and conclusive study, the test item 10-HSA was readily biodegradable after 19 days of exposure to domestic activated sludge under the conditions of the CO2 Evolution (Modified Sturm) Test performed according to the OECD Guideline for Testing of Chemicals, No. 301 B (1992), the Method C.4-C of Commission Regulation (EC) No 440/2008 and the US EPA OPPTS 835.3110 Ready Biodegradability, Paragraph (m) (1998).

Executive summary:

The test item 10-HSA was investigated for its ready biodegradability in a 28 Day CO2 Evolution (Modified Sturm) Test according to the OECD Guideline for Testing of Chemicals, No. 301 B (1992), the Method C.4-C of Commission Regulation (EC) No 440/2008 and the US EPA OPPTS 835.3110 Ready Biodegradability, Paragraph (m) (1998).

In accordance with the test guidelines the test was ended after 19 days since the biodegradation curves of the test item had reached a plateau over at least three determinations.

The test item 10-HSA was found to be biodegradable by 85 % under the test conditions within the 19 day exposure period.

The pass level for ready biodegradability, i.e. a CO2 formation of at least 60 % of the total organic carbon (TOC) in a 10 day window within the normally 28 day period of the test, was reached by exposure day 7 (62 %). In conclusion, 10-HSA was found to be readily biodegradable under the test conditions within 19 days.

In the toxicity control, containing both 10-HSA and the reference item sodium benzoate, biodegradation reached 78 % of the overall added TOC within 14 days of exposure. Thus, 10-HSA had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 20.8 mg/L.

In the procedure controls, average biodegradation of the reference item was 72 % by Exposure Day 5, and 90 % by Exposure Day 14, thus confirming suitability of the activated sludge (≥ 60 % degradation by Exposure Day 14). By the end of the test (Exposure Day 19), average biodegradation was 92 %. The validity criteria were fulfilled.