R-AMINR

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

OECD 209: 4 April 1984

Deviations:

no

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Seperate stock solutions of the test substance dissolved in double distilled water were made for each phase of this study and all solutions used within 24 hours of preparation. The concentration of all stock solutions was nominally 2 g/L.
- Range-finder: A single stock was prepared by dissolving 2 g of the substance into 1L double distilled water. Dissolution was achieved by subjecting the test substance and solvent to continuous ultrasonication for approximately 20 minutes. No traces of undissolved test substance remained after this treatment.
- Definitive: Three stocks of the substance were prepared in double distilled water on the day preceding the definitive assessment (all 2 g in 1L double distilled water). These were ultrasonicated as above for 25, 20 and 20 minutes respectively to achieve dissolution. The stocks were kept at ambient temperature overnight before use. The test substance remained completely dissolved in all three solutions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A sample of activated sludge was collected from one of the return sludge channels at Burley Menston sewage treatment works (Yorkshire water) whose waste-water catchment is predominantly domestic. On arrival at the lab the sludge was aerated by means of a compressed air supply delivered through a diffuser block.

The suspended solids concentration was determined by filtering a 25 mL sub-sample through a pre-weighed glass microfibre filter. The filter and retained solids were then dried at a nominal 110°C, re-weighed and the contribution made by the sludge solids determined by difference.

On the basis of the suspended solids determination, a portion of the sample was dilted with tap water to provide 10L inoculum whose suspended solids concentration was nominally 4 g/L. This inoculum was used in the range-finder test on the day it was collected. The inoculum was aerated continually. The inoculum was not acclimatised or adapted to the substance before exposure to the test substance.

The 7L inoculum remaining after the range-finder was maintained by feeding overnight with 100-fold OECD synthetic sewage concentrate at a rate of 50 mL/L on three consecutive days prior to the definitive assay. The inoculum was kept aerated continually throughout this period.

Immediately prior to the definitive experiment, the aeration was stopped and the sludge solids allowed to settle. The supernatant liquor was carefully decanted and the solids washed by restoring the volume with tap water. This preparation was re-settled and the supernatant discarded once more.

The pH of the residual solids was 7.04 (used without adjustment). The suspended solids concentration was re-determined as previously described, and on the basis of this determination, a portion of the sludge sample was diluted with tap water to provide 3L inoculum whose suspended solids concentration was nominally 4 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

Standard as per guideline

Post exposure observation period:

Not applicable

Hardness:

Not specified

Test temperature:

20 ± 2°C

pH:

pH of residual solids 7.04.

Dissolved oxygen:

Not measured overall

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Range-finder (nominal): control, 1, 10, 100 and 1000 mg/L
Definitive (nominal): Control, 375, 750, 1000, 1250 and 1500 mg/L

Details on test conditions:

TEST SYSTEM
- The substance was tested in duplicate over two concentration ranges in the two phases of the study. The reference inhibitor (3,5-DCP) was run in unreplicated preparations at concentrations of 5, 15 and 45 mg/L in both phases. Four controls were setup in both the range-finder and the definitive; two at the start and a pair at the end of the test series in both cases.

Incubations were begun sequentially at timed intervals, by adding the appropriate volume of test or reference stock, followed by inoculum, to preparations comprising 16 mL synthetic sewage concentrate and sufficient double distilled water to provide a total volume of 500 mL. Incubations entailed aerating the test preparations with a supply of compressed air delivered to each test vessel by a pipette and ran for three hours. At the end of the incubation dissolved oxygen (DO) measurements were made from a sub-sample of each preparation.

At the end of the 3 hour incubation period, a portion of the first test mixture was transferred to fill a clean 250 mL sample bottle containing a stir bar. The DO probe was inserted in the sample bottle, and the probe sealed against the bottle neck to ensure the sample could not re-oxygenate. At intervals of 15 minutes the procedure was repeated for each subsequent sample following the sequence in which the incubation began. A fresh sample bottle was used for each test mixture and the probe rinsed between samples.

Respiration rates were subsequently derived from the longest linear portion of each trace.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

999 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% Confidence limits 933 to 1069 mg/L

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None noted
- Effect concentrations exceeding solubility of substance in test medium: No
- Adsorption (e.g. of test material to the walls of the test container): None noted
- Blank controls oxygen uptake rate: 56.12 mgO2/L.h
- Coefficient of variation of oxygen uptake rate in control replicates: Not specified

Results with reference substance (positive control):

- Results with reference substance valid?: yes
- Relevant effect levels: 3 hour EC50 13 mg/L with 95% confidence limits 10 to 16 mg/L (between the allowed range of 5 to 30 mg/L)

Reported statistics and error estimates:

The applied concentrations of the substance and 3,5-DCP were first converted to Log10 and together with percentage inhibition data, subjected to linear regression analysis. The analysis was used to obtain regression coefficients (r) and to derive the effect concentration values.

A computer probit analysis program was also run to generate 95% confidence limits for effect concentrations of the substance and reference inhibitor.

The values derived from the computation program are used as the key values, with minor differences to the manually calculated values.

Respiration rate and inhibition data:

Treatment	Log10 conc.	Respiration rate (mg O2/L.h)	Mean	% Inhibition
Control	Na	57	56.12	na
	Na	57
	Na	55.06
	Na	55.42
375 mg/L substance	2.5740	53.06	52.99	6
		52.91
750 mg/L substance	2.8751	43.75	44.68	20
		45.60
1000 mg/L substance	3	26.66	26.26	53
		25.85
1250 mg/L substance	3.0969	17.43	17.26	69
		17.09
1500 mg/L substance	3.1761	12.63	12.58	78
		12.53
5 mg/L 3,5-DCP	0.6990	41.06	41.06	27
15 mg/L 3,5-DCP	1.1761	26.4	26.40	53
45 mg/L 3,5-DCP	1.6532	10.12	10.12	82

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, the 3 hour EC50 value to activated sludge based on respiration inhibition was 999 mg/L.

Description of key information

A reliable OECD guideline ASRIT study is available assessing the toxic potential of the substance to sewage treatment plant micro-organisms. This provided a 3 hour EC50 value of 999 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

999 mg/L