1-Naphthol, reaction products with formaldehyde

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 April 2005 to 03 June 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Preliminary solubility work showed that when the test material was dispersed in reverse osmosis purified deionised water it formed a navy blue solution. However when the test material was dispersed in culture medium it formed a brown dispersion with the test material having flocculated in clumps. It was therefore considered that the test material was reacting with inorganic salts in the culture medium forming an insoluble complex that resulted in the test material not forming a solution.
- Therefore for the purpose of the definitive study the test material was dissolved directly in deionised reverse osmosis water prior to dispersing in inoculated culture medium.
- An amount of test material (1000 mg) was dissolved in deionised reverse osmosis water with the aid of ultrasonication for approximately 10 minutes and the volume adjusted to 1 litre to give a 1000 mg/L stock solution. An aliquot (238.8 mL) of this stock solution was dispersed in inoculated culture medium and the volume adjusted to 3 litres to give a final concentration of 79.6 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the test material was inverted several times to ensure homogeneity of the solution.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: A mixed population of activated sewage sludge micro-organisms was obtained on 3 May 2005 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Laboratory culture: The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of 21 °C and used on the day of collection.
- Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper (previously rinsed three times with 20 mL deionised reverse osmosis water prior to drying in an oven) using a Buchner funnel. The filter paper was then dried in an oven at approximately 105 °C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids was equal to 3.0 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

other: Carbon

Initial conc.:

79.6 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: To 1 litre (final volume) of purified water (Reverse osmosis purified and deionised water, Elga Optima 15+ or Elga Purelab Option R-15 BP) was added 10 mL of Solution A and 1 mL each of Solutions B, C and D.
Solution A: 8.50 g/L KH2PO4, 21.75 g/L K2HPO4, 33.40 g/L Na2HPO4.2H2O and 0.50 g/L NH4Cl; pH = 7.4
Solution B: 27.50 g/L CaCl2
Solution C: 22.50 g/L MgSO4.7H2O
Solution D: 0.25 g/L FeCl3.6H2O
- An amount of test material (1 000 mg) was dissolved in deionised reverse osmosis water with the aid of ultrasonication for approximately 10 minutes and the volume adjusted to 1 litre to give a 1000 mg/L stock solution. An aliquot (238.8 mlL of this stock solution was dispersed in inoculated culture medium and the volume adjusted to 3 litres to give a final concentration of 79.6 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the test material was inverted several times to ensure homogeneity of the solution.
- Test temperature: 21 °C
- pH: 7.6 - 7.7
- pH adjusted: No. The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.
- Suspended solids concentration: 30 mg (ss)/ L (final concentration per vessel).
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 5 litre test culture vessels each containing 3 litres of solution.
- Number of culture flasks/concentration: 2; the test material, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/L.
- Method used to create aerobic conditions: Approximately 24 hours prior to addition of the test and standarde materials the vessels were filled with 2400 mL of culture medium and 30 mL of inoculum and aerated overnight. On Day 0 the test and reference materials were added and the volume in all the vessels adjusted to 3 L by the addition of culture medium. The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 mL/min stirred continuously by magnetic stirrer. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
- Measuring equipment: Hach HQ40d Flexi handheld pH meter, Shimadzu TOC-VCSH TOC analyser or a Shimadzu TOC-LCSH TOC analyser.
- Details of trap for CO2 and volatile organics if used: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.

SAMPLING
- Sampling frequency: Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.
- The samples taken on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 20, 22, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on Days 18 and 24 were stored at approximately -20 °C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed that the level of degradation of the test material did not significantly increase during this time and therefore additional analyses were considered to be unnecessary.
- On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
- Sampling method: The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser, Shimadzu TOC-VcsH TOC analyser and an Ionics 1555B TOC analyser. Samples (300, 50 or 40 µL) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air or nitrogen (oxygen free) as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

CONTROL AND BLANK SYSTEM
- Inoculum blank: A control, in duplicate, consisting of inoculated culture medium.
- Positive (procedure) control: The standard material, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/L. For the purposes of the test a standard material, sodium benzoate (C6HsCOONa), was used. An initial stock solution of 1000 mg/L was prepared by dissolving the standard material directly in culture medium with the aid of ultrasonication for approximately 5 minutes, and a 51.4 mL aliquot added to the test vessel to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the standard material was inverted several times to ensure homogeneity of the solution.
- Toxicity control: The test material plus the standard material in inoculated culture medium to give a final concentration of 20 mg carbon/L to act as a toxicity control ( one vessel only). For the purposes of the test a toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge micro­organisms used in the test. An aliquot (238.8 mL) of the test material stock solution was dispersed in inoculated culture medium along with an aliquot (51.4 mL) of the sodium benzoate stock solution. The volume was adjusted to 3 litres to give a final concentration of 79.6 mg test material/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

DISSOLVED ORGANIC CARBON ANALYSIS
- Samples (20 mL) were removed from the test material and toxicity control vessels on Day 0 prior to the addition of the test material in order to calculate the Inorganic Carbon/Total Carbon (IC/TC) ratio in the test media. The samples were filtered through Gelman 0.45 µm Acrocap filters (approximately 5 mL discarded) prior to DOC analysis.
- DOC analysis of the test material dispersions after dosing was not possible due to the insoluble nature of the test material in culture medium.
- On Days 0 and 28 samples (20 mL) were removed from the control and standard material vessels and filtered through Gelman 0.45 µm Acrocap filters (approximately 5 mL discarded) prior to DOC analysis.
- The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser. Samples (27 or 13 µL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.

STATISTICAL METHODS
-Calculation of Carbon Content
The theoretical amount of carbon present in the test material was calculated as follows:
[(No. of C atoms x mol wt of C) / mol wt of test material] x 100 %
[(44 x 12.011) / 714.68] x 100 = 73.95 %
The test material has a purity of 17 % and thus for a concentration of 10 mg C/L (a total of 238.8 mg) the total organic carbon present was 30 mg C.

The theoretical amount of carbon present in the reference material, sodim benzoate (C6H5COONa), was calculated as follows:
[(No. of C atoms x mol wt of C) / mol wt of sodium benzoate] x 100
[(7 x 12.0110)/144.11] x 100 = 58.34 %
Thus for a 10 mg C/L test concentration (a total of 51.4 mg) the total organic carbon present for sodium benzoate was 30 mg C.

-Percentage Biodegradation
The percentage biodegradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values into the following equation. The values of Replicates R1 and R2 are meaned for the inoculum control, test and reference materials before substitution into the following equation:
%ThCO2 (= % biodegradation) = [(mg IC in test flask – mg IC in control flask)/ mg TOC added as test material] x100 %
The percentage degradation from the results of the DOC analysis, is calculated from the equation below. Replicate values are corrected for the mean control value prior to calculation of percentage degradation.
Percentage degradation = [ 1 - (mg DOC in test flask on Day 28 / mg DOC in test flask on Day 0)] x 100 %

-Total CO2 Evolution
The total CO2 evolution in the control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation:
Total CO2 evolution = mg IC in control x (100 / %C of CO2) x (1 / test volume)
= mg IC in control x (100 / 27.29) x (1 / 3)

Statistical analysis was carried out using a Student's t-test on the Day 28 IC values for the control and test material vessels to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Reference substance:

benzoic acid, sodium salt

Test performance:

The validation criterion given in the OECD Test Guidelines were all satisfied.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

1

Sampling time:

28 d

Remarks on result:

other: St. dev. not reported.

Details on results:

- Percentage biodegradation values of the test and standard materials and the toxicity control are given in Table 1.
- The total CO2 evolution in the control vessels on Day 28 was 19.29 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
- The IC/TC ratio of the test material suspension in the mineral medium at the start of the test was below 5 % 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.
- The test material attained 1 % degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
- The slight increase in the degradation rate for the test material seen between days 10 -14 was due to mean inorganic carbon values for the replicate test material vessels increasing at a faster rate than the replicate control vessels. After Day 14 the degradation value in the test material vessels decreased. This was due to the inorganic carbon values in the replicate control vessels 'catching up' with those in the replicate test material vessels. It is not known why the inorganic carbon value in the control vessels lagged behind the test material vessels but it may be considered to be due to biological variation between the control and test material vessels.
- Statistical analysis of the Day 28 IC values for the control and test material vessels was carried out using a Student's t-test. There were no statistically significant differences (P≥ 0.05) between the control and test material Day 28 IC values. The test material was therefore considered not to have exerted a toxic effect on the sewage sludge micro-organisms used in the test.
- The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of control replicate R2 and the toxicity control vessel. These increases were considered to be due to C02 present in solution being driven off by the addition of hydrochloric acid on Day 28 and resulted in an increase in the percentage degradation value for the test material from 1 % on Day 28 to 2 % on Day 29.
- The toxicity control attained 39 % degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test. The decrease in inorganic carbon in the toxicity control and control replicate R2 first absorber vessels coupled with the increase in inorganic carbon within control replicate R1 on Day 29 resulted in a decrease in the percentage degradation value for the toxicity control from 39 % on Day 28 to 37 % on Day 29.
- Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of C02 into the second absorber vessels occurred.
- Analysis of the test media taken from the standard material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), gave percentage degradation values of 102 and 98 % respectively for Replicates R1 and R2. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence C02 evolution occurring. Degradation values in excess of 100 % were considered to be due to sampling/analytical variation.
- Observations made throughout the test period showed the contents of the control vessels to be light brown dispersions and the contents of the standard material vessels were light brown dispersions with no undissolved standard material visible. The contents of the test material vessels were light brown dispersions with flocculated clumps of test material visible. The contents of the toxicity control vessel was a light brown dispersion with no undissolved standard material visible, however flocculated clumps of test material were observed.

Results with reference substance:

Sodium benzoate attained 80 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in inorganic carbon in the replicate standard material and control replicate R1 first absorber vessels on Day 29 coupled with the decrease in inorganic carbon within control replicate R2 on Day 29 resulted in an increase in the percentage degradation value for the standard material from 80 % on Day 28 to 82 % on Day 29.

Table 1: Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate	% Degradation Test Material	% Degradation Test Material plus Sodium Benzoate Toxicity Control
0	0	0	0
1	23	0	0
2	39	0	10
3	39	0	13
6	48	0	27
8	49	0	30
10	65	4	38
12	78	8	43
14	74	11	45
16	78	7	44
20	77	6	44
22	80	2	42
27	82	2	39
28	80	1	39
29*	82	2	37

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this study, the test material attained 1 % degradation after 28 days and therefore cannot be considered to be readily biodegradable.

Executive summary:

The ready biodegradability of the test material was investigated in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and EPA OPPTS 835.3110, under GLP conditions.

The ready biodegradability of the test material was determined in an aerobic aqueous medium in the CO2 evolution test.

The test material, at a concentration of 10 mg C/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at 21 °C for 28 days.

The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

Under the conditions of this study, the test material attained 1 % degradation after 28 days and therefore cannot be considered to be readily biodegradable.

Description of key information

Under the conditions of this study, the test material attained 1 % degradation after 28 days and therefore cannot be considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The ready biodegradability of the test material was investigated in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and EPA OPPTS 835.3110, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The ready biodegradability of the test material was determined in an aerobic aqueous medium in the CO2 evolution test.

The test material, at a concentration of 10 mg C/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at 21 °C for 28 days.

The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

Under the conditions of this study, the test material attained 1 % degradation after 28 days and therefore cannot be considered to be readily biodegradable.