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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-11-14 - 2009-02-25; experimental completion: 2008-11-14;
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to relevant guideline (OECD 209) and under GLP. It is well documented and the results are plausible.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
In contrast to the guideline, it was decided to use copper (II) sulphate pentahydrate instead of the prescribed 3,5-dichlorophenol. The usage of dichlorophenol is unsuitable for routine lab use.
GLP compliance:
yes (incl. certificate)
Remarks:
According to § 19b German chemical act and directive 88/320/EEC
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
Test substance concentrations: 10 - 32 - 100 - 320 - 1000 mg/L
Dispersion treatment: Agitation
2 control samples without test item were included, one at the beginning and one at the end of the test.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
TEST SYSTEM: Activated sludge
Reason for the selection: Non adapted activated sludge from the sewage plant at Sarstedt is well suited as it receives predominantly municipal sewage and hardly industrial chemical waste.
Origin: Municipal sewage treatment plant of D-31157 Sarstedt
Pretreatment: The sludge was washed twice with autoclaved tap water and adjusted corresponding to a dry weight of 4.0 g/L ± 10 %.
Date of receipt: 2008-11-13
Dry sludge concentration: 3.98 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
not applicable
Hardness:
not applicable
Test temperature:
21°C
pH:
pH-value of the activated sludge: 6.78
Dissolved oxygen:
not applicable
Salinity:
not applicable
Nominal and measured concentrations:
not applicable
Details on test conditions:
TEST METHOD: static
Test duration: 3h
Replicates: One per concentration and two per control
Test vessels: 500 mL Erlenmeyer flasks, DIN 12380
Nutrient solution: Synthetic waste water according to OECD-Guideline 209
Volume of the test medium: 500 mL

Composition of test medium:
200 mL inoculum
16 mL synthetic waste water
284 mL autoclaved tap water
Test item concentration (directly weighing)

Aeration: permanent
Test temperature: 18-22 °C

TYPE AND FREQUENCY OF MEASUREMENT AND OBSERVATIONS:
The pH-vaiue of the activated siudge was measured prior to testing and the temperature in the measuring cell once during the measurement.
The oxygen depletion of the control, reference and test item replicates was measured after 3 h incubation by means of oximeter and recorded for at least 3 minutes on a flat bed recorder.
The room temperature during the test was recorded continuously by a hygrothermograph.

Equipment: pH-meter, CORNING 240; Oximeter, WTW, Oxi 197 - S; Flat bed recorder, LINSEIS; Hygrothermograph, LUFFT; Analytical balance, SARTORIUS; Balance, EW 3000 - 2M, KERN;

COURSE OF THE STUDY : A range-finding test (non GLP) was carried out with three concentrations: 10 - 100 - 1000 mg/L.
Based on the results of the range-finding test the study was carried out with 5 concentrations within the range 10 - 1000 mg/L in a geometrical series with a dilution factor of 3.2. Two control replicates without test item were included.
A fresh sample of activated sludge taken from the sewage plant Sarstedt was used for the test procedure. lt was washed twice and adjusted with autoclaved tap water to a dry sludge concentration of 4.0 g/L ± 10 %.
The reference item was tested in three concentrations within the concentration range 58 - 180 mg/L with a dilution factor of 1.8.
The respiration rates of all samples were determined subsequently to an incubation period of 3 h.

EVALUATION: The distances recorded by a flat bed recorder (adjusted to 500 mV and 1 cm/min in y-direction) during 3 min measurement were determined for each test vessel.
5 mm on the recorder (x-direction) are equivalent to a decrease of 0.1 mg O2/L. That leads to the factor f = 0.02 mg O2/L•mm.
The respiration rate (R) according to the raw data was calculated by formula (1)
R = md • f • t (1)
R : respiration rate mgO2/(L•h)
md : measured distance mm/3min
f : 0.02 mg O2/L•mm
t : 20 [3min/h]
The inhibition of respiration rates was calculated according to the guideline.

The deviation of the control respiration rates was calculated in %.

STATISTICAL ANALYSIS: The EC values of the test item were calculated by sigmoidal dose-response regression using software GraphPad Prism4, GRAPHPAD SOFTWARE, INC. Calculation of the confidence intervals for the EC50-value were carried out using standard procedures. The EC50-value of the reference item was calculated with probit analysis using software SiGmAPLor (Windows), SPSS CORPORATION. Probit values were taken from WEBER (1986): Grundriß der biologischen Statistik, 9. Auflage.



Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a. MERCK; CAS RN: 7758-99-8; Lot A573590; Purity: 99.7 %; Expiry date: 2009-07-31; Test concentrations: 58 - 100 - 180 mg/L; Range of validity (EC50): 54 - 156 mg/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
22.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
132 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 60.3 - 331
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
760 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
A clear concentration-effect relationship had been found with the test item.



Results with reference substance (positive control):
In the reference test the EC50 -value for copper(II)-sulphate-pentahydrate was determined by probit analysis with:
EC50 = 110.8 mg/L
This is in the recommended confidence range of 54 - 156 mg/L.

Respiration Rates and Inhibition of Respiration of Test and Reference ItemConcentrations

 

 

Concentration
[mg/L]

Respiration rate
[mg O2/(L•h)]

Inhibition
[%]

Test item

1000

3.6

88

 

320

13.6

54

 

100

13.6

54

 

32

20.8

29

 

10

28.8

2

Control 1

28.0

Control 2

30.8

Reference item

180

7.6

74

 

100

16.8

43

 

58

23.2

21

 

Difference of the control respiration rates: 9 %

Validity criteria fulfilled:
yes
Conclusions:
In a respiration inhibition test with acitvated sludge from a municipal sewage treatment plant performed according to OECD 209 and GLP (reliability category 1) the test item had been found to inhibit microbial respration by half at a concentration of 132 mg/L (EC50, 95% CI 60.3 - 331 mg/L ). The EC20 and EC80 had been determined as 22.8 and 760 mg/L, respectively. No classification is resulting from this impact of the test item on microorganisms.
Executive summary:

A Respiration Inhibition Test according to OECD Guideline No. 209 was performed with the test item o-Chlorbenzaldehyd on 2008-11-14 at DR.U.NOACK-LABORATORIEN, D-31157 Sarstedt. Test system was activated sludge of the municipal treatment plant of D-31157 Sarstedt. The test was performed under static conditions with the nominal concentrations 10 - 32 -100 - 320 - 1000 mg/L. The respiration rates of control, reference and test item concentrations were measured after a contact time of three hours, and the inhibitory effects of the test and reference concentrations were determined in comparison to the control respiration rates. A clear concentration effect relationship could be shown. The inhibition ranged from 2 to 88 %. The EC20-, EC50- and EC80-values were calculated via sigmoidal dose-response analysis (Table 1).

Table 1: EC-values with Confidence Interval of o-Chlorbenzaldehyd

 

EC-values
[mg/L]

Confidence interval
P = 95 [%]
[mg/L]

EC20

22.8

-

EC50

132

60.3 — 331

EC80

760

-

 

In order to check the activity of the test system and the test conditions a reference test was carried out with copper-(II)-sulphate-pentahydrate as reference item and the reference toxicity was determined. The EC50- value for the reference item was 110.8 mg/L. Validity criteria of the test guideline were fulfilled.

Description of key information

In a respiration inhibition test with acitvated sludge from a municipal sewage treatment plant performed according to OECD 209 and GLP (reliability category 1), the following inhibitory concentrations had been found for 2-chlorobenzaldehyde:
EC20 = 22.8 mg/L
EC50 = 132 mg/L (95 CI 60.3 - 331)
EC80 = 760 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
132 mg/L

Additional information

In a respiration inhibition test with acitvated sludge from a municipal sewage treatment plant performed according to OECD 209 and GLP (reliability category 1) the test item had been found to inhibit microbial respration by half at a concentration of 132 mg/L (EC50, 95% CI 60.3 - 331 mg/L ). The EC20 and EC80 had been determined as 22.8 and 760 mg/L, respectively.

This is supported by results from less reliable, non-guideline studies:

The static acute toxicity test for 2-chlorobenzaldehyde with Tetrahymena pyriformis (non guideline, reliability 3) covered 40 hours, covering 8 to 9 cell cycles in controls. Population densitiy was determined spetrophotometrically at 540 nm. This resulted in a IGC50of 0,326 mM (45,75 mg/L) for 2-chlorobenzaldehyde.

The Pseudomonas sp. Shk1 bioluminescence assay (nonguideline, reliability 3) results in an EC50 (5 minutes) of 67 mg/L.

From the experimental data available, the activated sludge respiration inhibition test is preferred over single organisms test as it covers a population of diverse organisms.