Copper diformate

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:

2019-06-04 to 2019-07-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Copper diformate

EC Number:

208-865-8

EC Name:

Copper diformate

Cas Number:

544-19-4

Molecular formula:

CH2O2.1/2Cu

IUPAC Name:

copper diformate

Test material form:

solid: particulate/powder

Details on test material:

blue in color

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): Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Method of cultivation: The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present.
- Storage conditions: The washed sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection.
- Pretreatment: none
- Concentration of sludge: 30 mg dry solids per litre
- Initial cell/biomass concentration: The suspended solids concentration was 3.0 g/L prior to use.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

10 mg/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:
A: Potassium dihydrogenphosphate KH2PO4 8.50 g
Dipotassium hydrogenphosphate K2HPO4 21.75 g
Disodium hydrogenphosphate dihydrate Na2HPO4 * 2 H2O 33.40 g
Ammonium chloride NH4Cl 0.50 g
are dissolved in demineralised water and made up to 1 litre.

B: Calcium chloride CaCl2 27.50 g
is dissolved in demineralised water and made up to 1 litre.

C: Magnesium sulfate heptahydrate MgSO4 * 7H2O 22.5 g
is dissolved in demineralised water and made up to 1 litre.

D: Iron (III) chloride hexahydrate FeCl3 * 6H2O 0.25 g
is dissolved in demineralised water, stabilised with one drop of concentrated HCl and made up to 1 litre.

For preparation of the mineral medium 10 mL of solution (A) is mixed with 900 mL demineralised water, 1 mL each of solutions (B), (C) and (D) are added and the volume is made up to 1 litre.

- Additional substrate:
- Test temperature: 22 °C - 24 °C
- pH: 7.4 (day 0 post-adjustment) to 7.5 (day 28)
- pH adjusted: yes
- Suspended solids concentration: 2.1 g/L
- Continuous darkness: yes
- Other: dry solid concentration: 180 mL activated sludge was filled up to 3000 mL with 2820 mL mineral medium corresponding to 30 mg/L dry solids.

TEST SYSTEM
- Number of culture flasks/concentration: 2 vessels in the inoculated control, the procedure control plus the reference item and the test item. 1 vessel only for the test item plus the reference item
- Method used to create aerobic conditions: The test vessels are aerated by the passage of carbon dioxide-free air.
- Measuring equipment: IC measurement was performed with a total carbon analyser (either TOC-V or TOC-L Shimadzu) by conversion of an aqueous sample to CO2 by orthophosphoric acid or 2M HCl using zero grade air as the carrier gas.
- Test performed in sealed vessels
- Details of trap for CO2 and volatile organics if used: The CO2 produced by degradation was absorbed in two 500 mL Dreschel bottles each filled with 350 mL 0.05 M NaOH.

SAMPLING
- Sampling frequency: IC analysis samples on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 from the first CO2 absorber and days 0 and 29 for the second CO2 absorber.
- Sampling method: IC analysis: 2 mL samples from the first of two CO2-absorber flasks was sampled and the IC's were determined. On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. IC/TC: 30 mL samples from the inoculum control and test item vessels and filtered through 0.45 µm Gelman AcroCap fillters and the IC/TC were determined. Samples (50 µL) were injected into the TC and IC channels of the TOC analyzer.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

62

Sampling time:

28 d

Details on results:

- The degradation extent of the test substance at the end of the test was 62%.
- The degradation extent in the toxicity control was 74% within 14 days and 77% biodegradation after 28 days. The test substance had no inhibitory effect on the inoculum.
- Test control Sodium benzoate attained 88% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 85% biodegradation was attained.
TOC confirmation: TOC of the diluted sodium benzoate stock solution gave a result of 92% of nominal and therefore confirmed that it had been prepared correctly.

Any other information on results incl. tables

Table1            Inorganic Carbon Values on Each Analysis Occasion

Day	Inorganic Carbon (mg IC)
	Inoculum Control				Procedure Control				Test Item				Toxicity Control
	R1		R2		R1		R2		R1		R2		R1
	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2	Abs1	Abs2
0	1.40	1.40	1.40	1.40	1.40	1.40	1.40	1.40	1.40	1.40	1.40	1.40	1.40	1.40
2	6.26	-	6.61	-	23.90	-	24.71	-	5.92	-	5.45	-	16.70	-
6	12.92	-	12.92	-	35.52	-	36.56	-	21.22	-	16.03	-	40.71	-
8	13.76	-	14.68	-	34.86	-	37.27	-	31.19	-	25.69	-	41.39	-
10	16.42	-	15.73	-	40.36	-	38.88	-	36.36	-	29.75	-	47.65	-
14	20.06	-	20.06	-	48.05	-	44.65	-	39.21	-	39.21	-	64.49	-
21	25.46	-	23.66	-	50.59	-	53.63	-	45.97	-	48.11/42.36**	-	69.52	-
28	31.81	-	30.58	-	57.01	-	60.14	-	50.40	-	48.72	-	68.65/76.16**	-
29	29.28/31.51*	2.78	28.50/30.28*	2.78	54.11	2.78	58.78	2.78	50.32	2.78	48.76	2.78	77.05	2.78

R = Replicate

Abs = CO₂absorber vessel

-  = No value determined

*  = Result from re-analysis of sample as original result was deemed to be erroneous

** = Result from re-analysis of frozen sample as original result was deemed to be erroneous

Table 2. Percentage Biodegradation Values

Day           Biodegradation (%)  Procedure Control Test Item   Toxicity Control  0  0  0 0   2  60 0  17   6  77 19  46   8  73 47  45   10  78 57  53   14  88 64  74   21  92 65  75   28  91 61  75   29*  85 62  77

* Day 29 values corrected to include any carry-over of CO₂detected in Absorber 2

Validation Criteria

The total CO2 evolution in the inoculum control vessels on Day 28 was 38.11 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.

The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed a decrease in all replicate vessels with the exception of test item Replicate 2 and the toxicity control. This decrease was considered to be due to sampling/analytical variation.

The IC analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item attained 62% biodegradation after 28 days and satisfied the 10-day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B. The test item does contain copper which is naturally occurring and stable in the environment and this part of the test item was considered to have not biodegraded.

Executive summary:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium when exposed to sewage sludge microorganisms under defined conditions. Carbon dioxide (CO₂) evolution from the test item at a concentration of 10 mg carbon/L was followed by means of Inorganic Carbon (IC) analysis over a period of usually 28 days under defined conditions. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO₂Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)). Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes. The test item attained 62% biodegradation after 28 days and satisfied the 10-day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.