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Toxicity to microorganisms

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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 17 NOV 2004 to 26 NOV 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented study performed according to relevant guidelines and compliant to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
Reference substance used: copper (II) sulphate pentahydrate instead of 3,5-dichlorophenol. The range of validity (EC50 ± 3SD) of copper (II) sulfate pentahydrate is calculated from the mean of all reference tests from 1991 - 2004 (n = 227).
GLP compliance:
yes (incl. certificate)
Remarks:
according to § 19b Abs. 1 German Chemikaliengesetz and directive 88/320/EEC

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
The test item was weighed out directly into an Erlenmeyer flask,
filled with 250 mL autoclaved tap water and stirred for 24 h.
Dispersion treatment: Agitation
Test concentration: Limit-test at 1000 mg/L

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
TEST SYSTEM: Activated sludge
Reason for the selection of study system: Non adapted activated sludge from the sewage plant at Sarstedt is well suited as it comprises mostly domestic sewage and hardly industrial waste water.
Origin: Domestic sewage treatment plant of D-31157 Sarstedt
Pretreatment: The sludge was washed twice with autoclaved tap water and diluted corresponding to a dry weight of 4.0 g/L +/- 10 %.
Date of receipt: 2004-11-26
Dry sludge concentration: 3.87 g/L

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Test temperature:
18-22 deg C
pH:
6.94
Dissolved oxygen:
See table on measurements under "any other information on results including tables"!
Nominal and measured concentrations:
Limit test at nominal 1000 mg/L submission substance
Details on test conditions:
2 control samples without test item were included, one at the beginning and one at the end of the test.
TEST METHOD: Static
Test duration 3 h
Replicates: One per concentration and two per control
Test vessels: 500 mL Erlenmeyer flasks, DIN 12380
Volume of the study medium: 500 mL
Nutrient solution: Synthetic waste water according to OECD 209

Application:
200 mL inoculum
16 mL synthetic waste water
284 mL autoclaved tap water
Test item concentration (directly weighing)

Test temperature: 18 - 22 °C
Ventilation: Permanent

TYPE AND FREQUENCY OF MEASUREMENT and OBSERVATIONS:
The pH value of the activated sludge 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 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.

COURSE OF THE STUDY:
A range-finding test (NON-GLP-state) was carried out with three concentrations: 10 - 100 - 1000 mg/L.

Based on the results of the preliminary test the study was carried out with a limit test concentration of 1000 mg/L. 2 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. It was washed twice and diluted 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:
Determined are the distances recorded by a flat bed recorder (adjusted to 500 mV and 1 cm/min in y-direction) during 3 min measurement 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 x mm.
The respiration rate (R) according to the raw data was calculated in the following way:
R = md x f x t
R : respiration rate mgO2/Lxh
md : measured distance mm/3min
f : 0.02 mgO2/Lxmm
t : 20 [3min/h]
The inhibition of respiration rates were calculated by a standard method as outlined in OECD 209.
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.

Results and discussion

Effect concentrationsopen allclose all
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:
The test item was hardly soluble. After stirring for 24 h the test item was mostly dispersed. Only part of the test item floated on the surface or stuck to the side of the flask.
For further details see section "Any other information on results including tables".
Results with reference substance (positive control):
REFERENCE ITEM Copper (II) sulphate pentahydrate p.a. MERCK
CAS RN 7758-99-8
Lot A265990
Purity 99.9 %
Expiry date 2005-11-30
Test concentrations 58 - 100 - 180 mg/L
Range of validity (EC50) 53 - 155 mg/L

In the reference test the EC50 -value for copper(II)-sulphate-pentahydrate was determined by probit analysis with:
EC50 = 125 mg/L
This is in the recommended confidence range of 53 - 155 mg/L.
Reported statistics and error estimates:
The EC20- and EC50-values were calculated with probit analysis using software SIGMAPLOT (Windows) rel. 8.02, 2002, SPSS CORPORATION.
Probit values were taken from WEBER (1986).
Confidence intervals were calculated according to a standard procedure (BREITIG & TÜMPLING 1982).

Any other information on results incl. tables

Raw Data and Respiration Rates of Test and Reference Item Concentrations

 

Concentration
[mg/L]

Oxygen-
concentration*
[mgO
2/L]

Measured
distance**
[mm/3min]

Respiration rate
[mgO
2/L¿h]

Test item

1000

7.01

81

32.4

Control 1

-

4.84

73

29.2

Control 2

-

6.65

83

33.2

Reference item

180

9.97

21

8.4

 

100

8.89

47

18.8

 

58

7.84

73

29.2

 

* determined at the start of the 3 minute measurement

** 5 mm are related to 0.1 mgO2/L

Respiration Rates and Inhibition of Respiration of Test and Reference ItemConcentrations

 

Concentration
[mg/L]

Respiration rate
[mgO2/L¿h]

Inhibition
[%]

Test item

1000

32.4

-4

Control 1

-

29.2

-

Control 2

-

33.2

-

Reference item

180

8.4

73

 

100

18.8

40

 

58

29.2

6

 Difference of the control respiration rates: 12 %

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In a respiration inhibition test with activated sludge according to OECD 209 no respiration inhibition was observed at the nominal test item concentration of 1000 mg/L (limit test).
EC50 (3h) > 1000 mg/L (nominal)
NOEC (3h) = 1000 mg/L (nominal)
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 and compliant to GLP (reliability category 1) was performed for 3 h with the submission substance. Test system was activated sludge of a domestic treatment plant. The test was performed under static conditions with the nominal concentration 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. No inhibition was observed. 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 125 mg/L. Validity criteria of the test guideline were fulfilled. The submission substance is not toxic at concentrations up to 1000 mg/L to activated sludge of a domestic sewage treatment plant.