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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:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Principles of method if other than guideline:
The test guidlines were adjusted to meet the Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C11
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge
Details on inoculum:
Test system: Micro-organisms in activated sludge.
Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
Number of micro-organisms: Number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium.
Preparation of the sludge: The sludge was coarsely 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 (3.0 g/l of sludge, as used for the test). The pH was 7.3 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic medium was added per litre of activated sludge at the endof the collection day. The sludge was kept aerated at test temperature until use.

Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l

Rationale: Recognized by international guidelines as the recommended test system.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
18.2-21.3°C
pH:
7.0-7.4
Nominal and measured concentrations:
nominal concentrations: 0-10-32-100-320-1000 mg/L loading of CdTe
Details on test conditions:
Contact time: 3 hours, during which aeration and stirring took place.
Vessels: All glass open bottles/vessels.
Milli-RO / Milli-Q water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).
Synthetic medium (=sewage feed) 16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-Q water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

Air supply:Clean, oil-free air.
Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/l at 20°C) and to maintain the sludge flocs in suspension.
Test set up: The final test was a standard test that did not include a nitrification inhibitor or abiotic conditions
Performance of the test: The synthetic medium (16 ml) and an adequate amount of the test substance loading were mixed and made up to 250 ml with Milli-RO water in a 1 litre bottle. The pH was determined. Thereafter 250 ml activated sludge was added. This was the start of the test. The mixture was then aerated during the contact time, using a pipette as an aeration device.After the 3-hour contact time, the oxygen consumption was recorded for a period of 9 - 11 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature were determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations/loading rates and controls.
Oxygen recording: Determination of oxygen was performed with oxygen probes connected to a BlueBox, a multichannel measuring and controlling system.
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.
Remarks:
CdTe
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
CdTe
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Reported statistics and error estimates:
ECx
For the reference substance the percentage inhibition was plotted against the logarithm of the concentrations and the EC50 was determined using linear regression analysis.

For Cadmium Telluride no EC50 could be calculated because the test substance proved to be non-toxic (EC50 > a loading rate of 1000 mg/l).

NOEC estimation
The NOEC was based on statistical analysis of the data. Data obtained for the test concentrations were compared with those obtained for the control using TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman.

Final test -study results:

Flask

Loading rate (mg/l)

Temp. (°C)

pH before addition of sludge

pH after 3 h contact time

Respiration rate

 (mg O2/l h)

Respiration rate

(mg O2/g h)¹

% Inhibition respiration relative to the control (mean value)

Control

0

18.8-21.2

7.4-7.5

7.1-7.3

41

27

-

T 1

10

19.5-21.3

7.4-7.5

7.3-7.4

41

27

-1

T 2

32

19.0-20.2

7.3-7.4

7.3-7.4

37

25

9

T 3

100

19.2-20.1

7.3

7.2-7.3

40

26

3

T 4

320

19.3-19.8

7.4

7.3

34

22

17*

T 5

1000

18.2-19.8

7.3-7.4

7.0-7.2

33

22

18*

¹           The amount of suspended solids in the final test mixture was 1.5 g/l.

*      Statistically significant (Bonferroni t-Test:α=0.05 Toxstat).

Validity criteria fulfilled:
yes
Conclusions:
Test done according standard protocol.
Executive summary:

The purpose of the study was to evaluate the test substance for its ability to adversely affect aerobic microbial treatment plants, and if possible to determine the EC50 and/or NOEC. The study was performed according to standard protocols. The EC50 was > 1g CdTe/L and NOEC was 100 mg CdTe/L.

Description of key information

The influence of CdTe on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The EC50 derived was > 1 g CdTe/L. However, as Cd2+ ion determines ecotoxicity, the PNEC deriviation will be based on the Cd 2+ ion as determined in the RA (ECB 2007).

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

Additional information

Under the conditions of the activated sludge respiration inhibition test CdTe was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 100 mg/L (NOEC). The EC50 derived was > 1 g CdTe/L. However, as Cd2+ ion determines ecotoxicity, the PNEC deriviation for STP will be based on the Cd 2+ ion as determined in the RA (ECB 2007).