6-aminopenicillanic acid

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to current guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For the purpose of the test, the test material was dispersed directly in water. The following test concentrations were assigned to the test: 10, 32, 100, 320 and 1000 mg/L. An amount of test material (2000 mg) was dispersed in water with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 litre to give a 2000 mg/L stock dispersion from which serial dilutions were made to give 200 and 20 mg/L stock dispersions. An aliquot (250 mL) of the 20 mg/L stock dispersion was dispersed with synthetic sewage (16 mL), activated sewage sludge (200 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (80 and 250 mL) of the 200 mg/L stock dispersion and aliquots (80 and 250 mL) of the 2000 mg/L stock dispersion were used to prepare the test concentrations of 32, 100, 320 and 1000 mg/L respectively. The volumetric flasks containing the stock dispersions were inverted several times to ensure homogeneity of the stock dispersions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection.
- Preparation of inoculum for exposure: An aliquot (250 mL) of the 20 mg/L test material stock dispersion was dispersed with synthetic sewage (16 mL), activated sewage sludge (200 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (80 and 250 mL) of the 200 mg/L stock dispersion and aliquots (80 and 250 mL) of the 2000 mg/L stock dispersion were used to prepare the test concentrations of 32, 100, 320 and 1000 mg/L respectively. The volumetric flasks containing the stock dispersions were inverted several times to ensure homogeneity of the stock dispersions. For the controls, at time "0" 16 mL of synthetic sewage was diluted to 300 mL with water and 200 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 – 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. A second control was later prepared.
- Suspended solids: The suspended solids concentration was equal to 3.9 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Total hardness of approximately 140 mg/L as CaCO3

Test temperature:

21 ± 1°C.

pH:

The pH of the sample was 7.3 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter

Dissolved oxygen:

Dissolved oxygen content is tabulated in the results

Nominal and measured concentrations:

Nominal concentrations of 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed
- Aeration: The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately
0.5 – 1 litre per minute.
- No. of vessels per concentration (replicates): 1 for each test material concentration and 1 for each reference material concentraton
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
Water parameters are listed in table 2 in the attached pdf Appendix I

OTHER TEST CONDITIONS
- Light intensity: The test was conducted under normal laboratory lighting

EFFECT PARAMETERS MEASURED : As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/L and 2.5 mg O2/:). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (Sigma-Aldrich Batch No. 04621CJ) 3.2, 10 and 32 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

590 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Any observations:
Observations made at 0 hours prior to the addition of activated sewage sludge and synthetic sewage showed that the test concentrations of 10, 32, 100, 320 and 1000 mg/L contained a clear colourless solution.
Observations made after 30 minutes contact time for the test concentration of 10, 32, 100, 320 and 1000 mg/L showed the vessels to contain a dark brown dispersion with no undissolved test material visible.
After 3 hours contact time the test concentrations of 10, 32, 100, 320 and 1000 mg/L all contained dark brown dispersions with no undissolved test material visible.

Results with reference substance (positive control):

- Relevant effect levels: EC20 – 1.9 mg/L at 3 hours
EC50 – 6.8 mg/L at 3 hours (95 % confidence limits 5.1-9.0 mg/L)
EC80 – 24 mg/L
NOEC – 1.5 mg/L

Reported statistics and error estimates:

Inhibition of respiration by the test and reference materials was expressed as a percentage of the two control respiration rates.
% inhibition = (-1(2RS/(RC1 + RC2)) x 100
RS = oxygen consumption rate for test or reference sample
RC1 + RC2 = oxygen consumption rates for controls 1 and 2
The percentage inhibition values were plotted against concentration, a line fitted using the Xlfit software package (IDBS) and the EC15, EC20, EC50 and EC80 values for the reference material and the EC15 and EC20 values for the test material determined from the equation for the fitted line. The EC50 and EC80 values for the test material were determined by inspection of the inhibition of respiration rate data. 95% confidence limits were calculated for the EC50 value for the reference material only using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949). The No Observed Effect Concentration (NOEC) was taken as being the EC15 value. The results of the study are considered valid if (i) the two control respiration rates are within 15% of each other and (ii) the EC50 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 5 to 30 mg/L.

Table3: Oxygen Consumption Rates and Percentage Inhibition Values in the Definitive Test after 3 Hours Contact Time

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2Reading (mg O2/L)	O2Consumption Rates (mg O2/L/min)	% Inhibition
Control	R1	6.6	10	1.9	0.47	-
	R2	6.1	8	2.3	0.48	-
Test Material	10	5.9	9	1.6	0.48	[1]
	32	5.8	9	1.6	0.47	1
	100	6.1	8	2.4	0.46	3
	320	6.4	8	2.8	0.45	5
	1000	7.2	10	4.2	0.30	37
3,5-dichlorophenol	3.2	7.3	10	4.0	0.33	31
	10	7.7	10	5.9	0.18	62
	32	8.3	10	7.6	0.07	85
R1-R2= Replicates 1 and 2

 

Table4 pH Values of the Test Preparations at the Start and End of the Exposure Period

Nominal Concentration (mg/l)		pH
		0 Hours	3 Hours
Control	R1	7.4	7.8
	R2	7.5	7.8
Test Material	10	7.5	7.8
	32	7.5	7.8
	100	7.3	7.8
	320	6.6	7.6
	1000	5.3	6.2
3,5-dichlorophenol	3.2	7.5	8.1
	10	7.5	8.1
	32	7.5	8.2
R1-R2= Replicates 1 and 2

 

Table5: Observations on the Test Preparations throughout the Test Period

Nominal Concentration (mg/l)		Observations on Test Preparations
		0 Hours	30 Minutes Contact Time	3 Hours Contact Time
Control	R1	Dark brown dispersion	Dark brown dispersion	Dark brown dispersion
	R2	Dark brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Material	10	Clear colourless solution*	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	32	Clear colourless solution*	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	100	Clear colourless solution*	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	320	Clear colourless solution*	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
	1000	Clear colourless solution*	Dark brown dispersion, no undissolved test material visible	Dark brown dispersion, no undissolved test material visible
3,5-dichlorophenol	3.2	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
	10	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
	32	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
R1-R2= Replicates 1 and 2 * = observations made prior to the addition of activated sewage sludge and synthetic sewage

Validity criteria fulfilled:

yes

Remarks:

The results of the study met the criteria set out in the guidelines.

Conclusions:

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 590 mg/L.

Description of key information

Toxicity to microorganisms of 6-Aminopenicillanic acid was determined by OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test).

Additional information

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 590 mg/L.