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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:
31 JULY 2018 to 30 OCTOBER 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
Commission Regulation (EC) No. 440/2008, Method C.4-C, adopted 30. May 2008
“CO2-Evolution-Test”
Deviations:
yes
Remarks:
The carbon analyser was not calibrated monthly as stated in the study plan. Because quality control samples were measured daily, this can be considered as uncritical concerning the outcome of the study.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
 OECD Guidelines for the Testing of Chemicals, Part 301 B, adopted 17. Jul. 1992
“CO2-Evolution-Test (Modified STURM Test)“
 Commission Regulation (EC) No. 440/2008, Method C.4-C, adopted 30. May 2008
“CO2-Evolution-Test”
Deviations:
yes
Remarks:
The carbon analyser was not calibrated monthly as stated in the study plan. Because quality control samples were measured daily, this can be considered as uncritical concerning the outcome of the study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
- Laboratory culture: The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, 67435 NW-Lachen-Speyerdorf.
- Method of cultivation: The sludge was filtrated, washed with test medium (2x) and re-suspended in test medium. It was then aerated until use.
- Storage conditions: It was then aerated until use.
- Storage length: 2 days (Date of collection: 28. Sep. 2018, Date of use 1. Oct. 2018)
- Preparation of inoculum for exposure: None
- Pretreatment: The sludge was filtrated, washed with test medium (2x) and re-suspended in test medium. It was then aerated until use. The dry matter was determined to contain 4940 mg of suspended solids/L.
- Concentration of sludge: The dry matter was determined to contain 4940 mg of suspended solids/L.
- Initial cell/biomass concentration: The carbon content of 74.51 % was calculated by molecular formula given by the sponsor. The test item was weighed directly, the amounts were calculated from the carbon content of the test item. The test was performed with a nominal start concentration of 20 mg organic carbon/L of the
test item.
- Water filtered: yes
- Type and size of filter used, if any:
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:The medium was freshly prepared. 10 mL of solution a were mixed with 800 mL water, then 1 mL of solutions b, c and d were added and filled up to 1 L with H2O demin (volumes were adapted to final volume needed in the test).
Composition:
Solution a 10 mL
Solution b 1 mL
Solution c 1 mL
Solution d 1 mL
H2O demin. ad 1000 mL

Solution a
Potassium dihydrogen phosphate (KH2PO4) 8.5 g
Dipotassium hydrogen phosphate (K2HPO4) 21.75 g
Disodiumhydrogen phosphate dihydrate (Na2HPO4*2H2O) 33.4 g
Ammonium chloride (NH4Cl) 0.5 g
H2O demin. ad 1000 mL
The pH was 7.4.

Solution b
Calcium chloride (CaCl2) 27.5 g
H2O demin. ad 1000 mL

Solution c
Magnesium sulphate heptahydrate (MgSO4*7H2O) 22.5 g
H2O demin. ad 1000 mL

Solution d
Iron(III) chloride hexahydrate (FeCl3*6H2O) 0.25 g
Di-sodium-ethylene diaminetetraacetate dihydrate (Na2EDTA*2H2O) 0.4 g
H2O demin ad 1000 mL

- Additional substrate: none
- Solubilising agent (type and concentration if used): none
- Test temperature: 20.1 – 22.8 °C without direct lighting
- pH: NA
- pH adjusted: no
- CEC (meq/100 g):
- Aeration of dilution water: yes
- Suspended solids concentration: The dry matter was determined to contain 4940 mg of suspended solids/L
- Continuous darkness: yes
- Other:

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration:
Flask volume 1500 mL
Apparatus blanks 2, containing mineral medium only
Blank Controls 2, containing mineral medium and inoculum
Positive control flasks 2, containing positive control, mineral medium and inoculum
Test flasks 2, containing test item, mineral medium and inoculum
Abiotic control 1, containing test item, mineral medium and HgCl2
Toxicity control 1, containing test item, positive control, mineral medium and inoculum
- Method used to create aerobic conditions: The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, moistened air. The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 M NaOH. To control the absence of CO2, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels.
- Method used to create anaerobic conditions:
- Measuring equipment: the carbon analyser TOC multi N/C 2100S, Analytik Jena.
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used: The emitted CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels. The initial IC value of the 0.25 M NaOH was separately determined in each flask.
- Other:

SAMPLING
- Sampling frequency: From each front scrubber flask, 9 samples were taken in order to determine the emitted CO2 (on day 0, 3, 7, 9, 11, 14, 17, 23 and 29). On day 28, 5 mL HCl 2 M was added to each test flask in order to drive off dissolved CO2
- Sampling method: The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2
- Sterility check if applicable:
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: lank Controls 2, containing mineral medium and inoculum
- Abiotic sterile control:
- Toxicity control: oxicity control 1, containing test item, positive control, mineral medium and inoculum
- Other:

STATISTICAL METHODS:
The percentage biodegradation in the test flasks was calculated from:
% degradation = (((emitted C (Test) in mg/L)-(Mean emitted C (Controsl) in mg/L))/(added C in mg/L))*100

Degradation in positive control and toxicity flasks was calculated analogously.
Abiotic degradation was calculated from:
% degradation = ((emitted C (abiotic) in mg/L))/(added C in mg/L))*100
Reference substance
Reference substance:
aniline

Results and discussion

% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 8
St. dev.:
6.7
Sampling time:
28 d
Remarks on result:
other: not readily biodegradable and not ultimately biodegradable, within 28 days
Details on results:
Day 3: 1.1%
Day 7: 2.8%
Day 9: 2.8%
Day 11: 4.1%
Day 14: 5.8%
Day 17: 8.7%
Day 23: 8.3%
DAy 29: 7.7%

BOD5 / COD results

Results with reference substance:
The test item 1,4-bis(isopropylamino)anthraquinone is considered as “not readily biodegradable“.
 The degree of biodegradation reached 8 % after 28 days.
 The 10-day-window was not detected.
 Degradation missed 60 % within 28 days, too. Therefore, the test item 1,4-
bis(isopropylamino)anthraquinone is considered as “not ultimately biodegradable,
within 28 days”, as well.
 Abiotic degradation was not observed.

Any other information on results incl. tables

In the following table, the percentage biodegradation is presented:
Table 8.3-b Degradation values in %








































































































Day Positive
Control
1
Positive
Control
2
Positive
Control
Mean
Test 1 Test 2 Test
Mean
Abiotic
Control
Toxicity
Control
3 0.6 -1.5 -0.5 1.1 -0.3 0.4 1.1 2.2
7 34.9 42.0 38.5 1.6 4.0 2.8 0.6 25.0
9 51.6 60.0 55.8 0.8 4.8 2.8 0.2 34.3
11 70.3 70.3 70.3 3.2 5.0 4.1 0.0 39.9
14 81.9 80.5 81.2 5.9 5.7 5.8 0.1 44.0
17 86.5 82.5 84.5 8.9 8.6 8.7 -0.2 46.7
23 91.8 84.9 88.3 5.5 11.0 8.3 -1.1 46.7
29 95.1 83.4 89.2 4.4 11.1 7.7 -2.2 49.3

Because the values of day 29 are the sum of the IC values in both scrubber flasks, an
increase (IC values in flasks B of the test higher than in those of the control) or a decrease
(IC values in flasks B of the test lower than in those of the control) of degradation can be
observed.
As the measured IC values in the abiotic control are very low, measurement uncertainties
lead to negative degradation values while in fact no degradation has taken place.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
 The test item 1,4-bis(isopropylamino)anthraquinone is considered as “not readily bi
odegradable“.
 The degree of biodegradation reached 8 % after 28 days.
 The 10-day-window was not detected.
 Degradation missed 60 % within 28 days, too. Therefore, the test item 1,4-
bis(isopropylamino)anthraquinone is considered as “not ultimately biodegradable,
within 28 days”, as well.
 Abiotic degradation was not observed.