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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
Study period:
09 Nov 2017 - 10 Nov 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
22 July 2010
GLP compliance:
yes
Specific details on test material used for the study:
Identification: Cedarwood Texas oil crude
Appearance: Yellow liquid (determined by Charles River Den Bosch)
Batch: LS160616
purity/composition: UVCB
Test item storage: At room temperature
Stable under storage conditions: until 15 June 2018 (expiry date)
Specific gravity/density: 0.954 – 0.966 at 20°C
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The batch of Cedarwood Texas oil crude tested was a yellow liquid (UVCB) with a purity of 100%. No correction was made for the purity or composition of the test item. Since the test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L, aliquots corresponding to the test concentrations were pipetted into the dark brown test-bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). The test item – Milli-RO water mixtures were magnetically stirred for a short period and subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added, resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's- Hertogenbosch, The Netherlands, receiving predominantly domestic sewage
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. The pH was 7.6 on the day of testing.
- Pretreatment: 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 end of the collection day. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration: A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids, i.e. 3.0 g/L of sludge. The amount of suspended solids in the final test mixture was 1.5 g/L.
Test type:
static
Water media type:
freshwater
Remarks:
water Tap water purified by reverse osmosis (Millipore Corp.,Bedford, Mass., USA)
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3-hour exposure period.
Test temperature:
20 - 22°C
pH:
T 0hr control: 7.3
T 3hr control: 7.0-7.6

T 0hr test item: 7.3 - 7.4
T 3hr test item: 7.1-7.2

T 0hr 3,5 DCP: 7.3-7.4
T 3hr 3,5 DCP: 7.9-8.0
Dissolved oxygen:
>60-70% of air saturation at test start
Nominal and measured concentrations:
Test substance
Nominal: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: all glass open bottles/vessels
- Total fill volume: 500 mL (250 mL test solution (incl. synthetic medium) + 250 mL activated sludge in test medium)
- Aeration: yes (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)
- No. of vessels per concentration (replicates): 1 replicate for the lower conentrations and 3 for the highest concentration
- No. of vessels per control (replicates): 6
- No. of vessels per positive control (replicates): 4
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 1.5 g/L sludge
- Nutrients provided for bacteria: synthetic medium acc. to OECD 209
- Nitrification inhibitor used : no

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Details on termination of incubation: After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
- Synthetic medium:
16 g peptone (=sewage feed)
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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen uptake

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: loading rates of 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes; The combined limit/range-finding test showed no inhibition, 2% inhibition and an average of 21% inhibition of the respiration rate at a concentration 10, 100 and 1000 mg/L, respectively.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
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
Details on results:
Inhibition of the Respiration Rate:
The combined limit/range-finding test showed no inhibition, 2% inhibition and an average of 21% inhibition of the respiration rate at a concentration 10, 100 and 1000 mg/L, respectively. For Cedarwood Texas oil crude, no EC50-value could be calculated because effects were below 50% (EC50 > 1000 mg/L).
In the present test Cedarwood Texas oil crude was slightly, but significantly toxic (21% inhibition) to waste water (activated sludge) bacteria at 1000 mg/L. Therefore, a NOEC could not be determined. There was no oxygen uptake from abiotic processes.

Acceptability of the test:
1. The mean control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (23 mg oxygen per one gram of activated sludge).
The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test (6%).

2. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (5 mg/L).
Results with reference substance (positive control):
The batch of activated sludge was tested for sensitivity with the reference item 3,5- dichlorophenol, and showed normal sensitivity.
EC50 = 4.9 mg/L ( 95% Cl: 3.6 - 6.3) (falls within the OECD reported range for total respiration, i.e. 2-25 mg/L)
Reported statistics and error estimates:
Evaluation was based on the inhibition of the total respiration.

ECx and ECx:
For the reference item calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.

NOEC determination:
An effect was considered to be significant if statistical analysis of the data obtained for the concentrations compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Two-sample t-test Procedure, α=0.05, one-sided, smaller). Calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Results: Respiration Rate/Inhibition

Replicate

Concentration (T)

Concentration (R)

(mg/L)

Respiration rate

% Inhibition

respiration rate

(mean value)

(mg O2/L.h)

(mg O2/g.h)¹

C 1

0

37.94

25.29

 

C 2

0

33.44

22.29

 

C 3

0

34.99

23.33

 

C 4

0

35.11

23.41

 

C 5

0

32.28

21.52

 

C 6

0

35.61

23.74

 

C Mean

 

34.90

23.26 (RC)

 

SD

 

1.94

1.29

 

CV (%)

 

6

6

 

 

 

 

 

R 1

1.0

31.04

20.69

11.05

R 2

3.2

20.18

13.45

42.17

R 3

10

11.75

7.83

66.33

R 4

32

3.99

2.66

88.57

 

 

 

 

 

T 1

10

36.65

24.43

-5.03

T 2

100

34.18

22.79

2.05

T 3a

1000

26.73

17.82

23.40

T 3b

1000

27.69

18.46

20.65

T 3c

1000

28.69

19.13

17.78

T3 Mean

27.70

18.47 (RT)*

20.61 (IT)

 

 

 

 

 

TA

1000

0.00#

0.00#

100.00

 

C:         Control

R:         Reference item, 3,5-dichlorophenol           

T:         Test item, Cedarwood Texas oil crude        

TA:      Abiotic control of Cedarwood Texas oil crude        

SD:      Standard deviation           

CV:      Coefficient of variation                  

RT:        Total respiration with Cedarwood Texas oil crude

RC:        Total respiration in the control

IT:          % inhibition of total respiration relative to RC

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

*            Statistically significantly different compared to control       

#            No respiration, therefore expressed as 0 mg O2/L.h

Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
Cedarwood Texas oil crude was slightly, but significantly toxic (21% inhibition) to waste water bacteria (activated sludge) at a concentration of 1000 mg/L. The EC50 was above 1000 mg/L.
Executive summary:

A study was performed to assess the effect of Cedarwood Texas oil crude on the respiration of activated sewage sludge according to OECD 209 and GLP guidelines. In a combined limit/range finding test, micro-organisms in activated sludge (in test medium) were exposed to test concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for 3 hours. Optimal contact between the test item and test medium was ensured by applying continuous stirring and aeration during that period. In parallel, an untreated control (6 replicates), reference substance (3,5 -dichlorophenol, 4 replicates) and abiotic control (1 replicate) were tested. After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes (no aeration but continuous stirring).The study met the acceptability criteria prescribed by the study plan and was considered valid. There was no oxygen uptake from abiotic processes and the reference item 3,5- dichlorophenol showed normal sensitivity. Cedarwood Texas oil crude was slightly, but significantly toxic (21% inhibition) to waste water (activated sludge) bacteria at a concentration of 1000 mg/L. Therefore, a NOEC could not be determined. The EC50 was determined to be above 1000 mg/L.

 

Description of key information

A study was performed to assess the effect of Cedarwood Texas oil crude on the respiration of activated sewage sludge according to OECD 209 and GLP guidelines. In a combined limit/range finding test, micro-organisms in activated sludge (in test medium) were exposed to test concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for 3 hours. Optimal contact between the test item and test medium was ensured by applying continuous stirring and aeration during that period. In parallel, an untreated control (6 replicates), reference substance (3,5 -dichlorophenol, 4 replicates) and abiotic control (1 replicate) were tested. After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes (no aeration but continuous stirring).The study met the acceptability criteria prescribed by the study plan and was considered valid. There was no oxygen uptake from abiotic processes and the reference item 3,5- dichlorophenol showed normal sensitivity. Cedarwood Texas oil crude was slightly, but significantly toxic (21% inhibition) to waste water (activated sludge) bacteria at a concentration of 1000 mg/L. Therefore, a NOEC could not be determined. The EC50 was determined to be above 1000 mg/L.

 

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

The EC50 is > 1000 mg/L