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Toxicity to microorganisms

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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:
other: GLP guideline study (deviations to guideline do not affect the study integrity)
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
One measurement of temperature was started without sludge (30 min later the sludge was added to the Milli-RO and synthetic medium). In the range finding test the range of one temperature measurement was slightly above 20 ± 2 °C.
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Health Care Inspectorate, Ministry of Health, Welfare and Sport, Den Haag, The Netherlands
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
- Method: A stock solution of 10 g/L was prepared by adding 5 g test substance to 500 mL of Milli-RO water. In the final test the test substance was crushed prior to the preparation of the stock solution. Magnetic stirring for 29 min was applied. Corresponding volumes of the clear light yellow stock solution were then added to the test media.
- Eluate: no
- Differential loading: yes
- Controls: yes, blank control
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Preparation of inoculum for exposure: Activated sludge was obtained from the treatment plant ‘Watershap Aa en Maas’, s-Hertogenbosch, The Netherlands, which treats predominantly domestic sewage. The sludge was sieved (1 mm), washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105 °C to determine the amount of suspended solids (3 g/L of sludge). The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (= sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19.7 - 21.1 °C
pH:
before addition of sludge: 7.2 - 9.0
after 3 h contact time: 7.7 - 8.3
Nominal and measured concentrations:
Nominal: control, 46, 100, 220, 460 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass bottles
- Type: Open
- Aeration: Adjusted in such a way that the dissolved oxygen concentration at the start was > 60-70% saturation (60% of air saturation is > 5 mg/L at 20 °C)
- No. of vessels per concentration: 5
- No. of vessels per control: 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Millipore Corp., Bedford, USA)
- Culture medium different from test medium: No, according to OECD guideline 209
- Intervals of water quality measurement: The pH and temperature were measured at the start and end of the test.

EFFECT PARAMETERS MEASURED: The respiration rate was measured after a test period of 3 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
Range finding study:
- Test concentrations: control, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: No inhibition of respiration at 10 and 100 mg/L, 24% inhibition at 1000 mg/L (statistically significant)
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- No statistically significant inhibition of the respiration rate of the sludge was recorded at any of the concentrations.
- In the combined limit/range-finding test a significant effect was observed at 1000 mg/L based on 3 replicates. Considering the higher number of replicates in the final test, the results of the latter one were therefore used for determination of the effect values.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was in the accepted range of 2-25 mg/L for total respiration (EC50 = 3.4 mg/L).
Reported statistics and error estimates:
- Student t-test for homogenous variance with Bonferroni-Holm adjustment (α = 0.05 one sided smaller) to determine significance of inhibition of the respiration rate.

Table 1: Preparation of test solutions

Treatment

Test / reference substance

Synthetic sewage [mL]

Made up to 250 mL with Milli-RO water

Sludge [mL]

Blank control

no

16

yes

250

Test substance

yes

16

yes

250

Reference substance

yes

16

yes

250

 

Table 2: Study results (pH, respiration rate and % inhibition)

Treatment

Concentration (mg/L)

Respiration rate (mg O2/L/h)

Respiration rate (mg O2/g/h) *

% inhibition respiration relative to control (mean)

Control (mean)

-

55

37

-

Reference substance

2.0

34

23

39

5.0

22

15

60

12

15

10

73

Test substance (mean)

46

54

36

2

100

59

39

-7

220

53

35

5

460

54

36

2

1000

48

32

14

* The amount of suspended solids in the final mixture was 1.5 g/L

Description of key information

NOEC (3 h): ≥ 1000 mg/L for activated sludge microorganisms (OECD 209)

Key value for chemical safety assessment

Additional information

The toxicity of the test substance to microorganisms was investigated in a respiration inhibition study according to OECD 209 under GLP conditions (Tosoh Corporation, 2014). The main test was performed based on the result of a preceding combined limit/range-finding test. Five concentrations, ranging from 46 to 1000 mg/L, were tested. No statistically significant inhibition of the respiration rate of the sludge was recorded up to the highest concentration of 1000 mg/L. The test with the reference substance confirmed normal sensitivity of the activated sludge. Therefore a NOEC (3 h) of ≥ 1000 mg/L for aquatic microorganisms has been determined.