[No public or meaningful name is available]

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

29 Aug - 04 Oct 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

1992

Deviations:

yes

Remarks:

See deviation within the section "Any other information on material and method incl. tables"

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ISO standard 10634

Version / remarks:

1995

Deviations:

yes

Remarks:

See deviation within the section "Any other information on material and method incl. tables"

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Vapour grown graphitic carbon fibre

Molecular formula:

C

IUPAC Name:

Vapour grown graphitic carbon fibre

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge from a municipal sewage treatment plant, 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, predominantly domestic sewage.
- Storage conditions: Mineral components, Milli-RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
- Storage length: Over night
- Preparation of inoculum for exposure: Sludge was sieved (1 mm) and washed with mineral medium.
- Concentration of sludge: 4.5 g/L in concentrated sludge (suspended soilds)

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

12 mg/L

Based on:

TOC

Remarks:

approxmately 13 mg/L test substance

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: According to the guideline; Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.
- Test temperature: 22.3 and 22.8 °C
- pH: 7.6 at test start: 7.55; at test end: 7.55 (blank control), 7.61-7.62 (test item)
- pH adjusted: yes
- Aeration of dilution water: Synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per sec (ca. 30-100 mL/min).
- Suspended solids concentration: 4.5 g/L in concentrated sludge (suspended soilds)
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured glass bottles
- Number of culture flasks/concentration: 2 replicates (with test item)
- Method used to create aerobic conditions: A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts.
- Measuring equipment: TOC analysis was performed using a Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu SSM-5000A (Solid Sample module for Total Organic Carbon Analyzer) (Shimadzu, Kyoto, Japan).
- Test performed in open system: No
- Details of trap for CO2: Emitted CO2 was trapped in 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2

SAMPLING
- Sampling frequency: Day 2, 5, 8, 12, 15, 19, 23, 29 (Biodegradation is ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28).
Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test item. Positive and toxicity control: at least 14 days.
- Sampling method: CO2 produced in each test bottle reacted with barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 bottles
- Toxicity control: 1 bottle
- Other: Reference: 1 bottle

Reference substance

Reference substance:

other: Sodium acetate

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (CO2 evolution)

Value:

3

Sampling time:

28 d

Remarks on result:

other: Vessel A

% Degradation 2

Parameter:

% degradation (CO2 evolution)

Value:

4

Sampling time:

28 d

Remarks on result:

other: Vessel B

Details on results:

In the toxicity control, biodegradation amounted to 43% within 14 days. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration due to a biodegradation >25%.

BOD5 / COD results

Results with reference substance:

Reference item was biodegraded by at least 60% (actual result: 82%) within 14 days.

Any other information on results incl. tables

Table 1: CO2 Production and Percentage Biodegradation of the Reference Item

Day	HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation 1) (%)
	Blank (mean)	Procedure control
2	48.95	40.28	8.67	9.5	9.5	11
5	46.95	20.24	26.71	29.4	38.9	46
8	44.21	28.89	15.32	16.8	55.8	65
12	41.59	36.31	5.28	5.8	61.6	72
15 2)	45.12	37.21	7.90	8.7	70.3	82

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of sodium acetate: 85.5 mg CO2/2L.

2): CO2 measured on day 15 is actually part of CO2 production of day 14, since microbial activity was ended on day 14 by addition of HCl.

Table 2: CO2 Production and Percentage Biodegradation of the Toxicity Control

Day	HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation 1) (%)
	Blank (mean)	Toxicity control
2	48.95	42.97	5.98	6.6	6.6	4
5	46.95	18.88	28.07	30.9	37.4	21
8	44.21	26.92	17.29	19.0	56.5	32
12	41.59	32.65	8.94	9.8	66.3	38
15 2)	45.12	37.21	7.90	8.7	75.0	43

1): Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test item and reference item: 174.5 mg CO2/2L (ThCO2 test item: 89.0 mg CO2/2L + ThCO2 sodium acetate: 85.5 mg CO2/2L).

2): CO2 measured on day 15 is actually part of CO2 production of day 14, since microbial activity was ended on day 14 by addition of HCl.

Table 3: Comparison of Biodegradation of the Test Item in Bottles A and B

Day	Biodegradation (%)
	Bottle A	Bottle B	Mean A and B	Δ A-B 1)
2	0	0	0	0
5	0	3	2	3
8	0	3	2	3
12	0	3	2	3
15	0	3	2	3
19	2	3	3	1
23	2	3	3	1
29 2)	2	4	3	2
29 2)	2	4	3	2
29 2)	3	4	4	1

1): Absolute difference in biodegradation between bottles A and B

2): Biodegradation is ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.

Table 4: Validity criteria for OECD 301 B.

Criterion from the guideline	Criterion from the guideline	Criterion from the guideline
Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.	≤3%	Yes
Percentage degradation of the reference compound has reached the pass levels by day 14.	82% within 14 days	Yes
The IC content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC.	< 5%	Yes
The total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.	29.2 mg CO2/L	Yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Interpretation of results:

not readily biodegradable