Registration Dossier

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 November 1993 to 01 July 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: As the test material was considered insoluble, no stock solution was prepared and the material was added directly to each test vessel by way of glass microscope cover slips.

The dilution water for this test was from the City of Franklin, Tennessee, water supply. The water was softened and dechlorinated prior to use. Dechlorination was confirmed with the DPD colorimetric method (APHA, 1989).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge with associated aerobic organisms were obtained from the Cottonwood Subdivision Wastewater Treatment Plant located in Franklin, Tennessee. This wastewater treatment plant serves only the residential subdivision and its community pool.

- Method of cultivation:
- Preparation of inoculum for exposure: One gallon of activated sludge was collected by the plant operator, picked up on the day of collection by WCC personnel and transported to the testing laboratory. Immediately upon arrival, the sludge was aerated with low-pressure, oil-free air.
- Pretreatment: The activated sludge organisms were fed a synthetic sewage feed (see below) at a rate of 50 mL per liter.

Synthetic Sewage Medium (per 1 L of solution)
- 16.0 g Peptone
- 11.0 g Beef Extract
- 3.0 g Urea
- 0.70 g NaCl
- 0.40 g CaC12 -2H2O
- 0.20 g MgSO4- 7H2O
- 2.80 g K2HPO4

- Initial biomass concentration:
Triplicate 4-mL samples of the mixed sludge were dried at 100 °C in a Fisher brand drying oven, until a constant weight was achieved. Based on these results the sludge was diluted to produce a dry weight per unit volume concentration of 4 g/L.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
The incubator maintained a constant temperature of 20 °C.
Nominal and measured concentrations:
The nominal test material concentrations were 1, 10, 100, 1 000, and 10 000 mg/L.
Details on test conditions:
TEST SYSTEM
- The testing system consisted of two units: 1) a system used to expose the sludge inoculum to the test material; and 2) a system to measure the rate of oxygen consumption of the sludge in a closed bottle.
- Test vessel: 1 L flint glass bottles
- Washing of glassware: All bottles and glassware used in testing were cleaned with soap, 10 % nitric acid, and acetone and WCC reagent grade water
- Aeration: Low-pressure oil-free air was delivered to each test vessel using a glass pipet. Air pressure was controlled by lines connected by brass needle valves.

TEST MEDIUM / WATER PARAMETERS
To measure the rate of oxygen consumption for the activated sludge after exposure to the test materials a Model 51 Yellow Springs Instruments (YSI) dissolved oxygen meter was used. The oxygen meter was fitted with a BOD bottle probe, with a standard sensitivity membrane and an external output to a Fisher Recordal (Model 5000) strip chart recorder. The strip chart recorder was set at the 100-mV range and run at 1 cm per minute. To measure the consumption of oxygen, a sub-sample of the exposed sludge was placed in a clean BOD bottle, the change in oxygen concentration over time was measured with the YSI meter and probe, and the results were recorded on the strip chart recorder.

OTHER TEST CONDITIONS
- Photoperiod: 3 hours of darkness

CONTROL FLASK
16 mL of the synthetic sewage feed, 284 mL of dechlorinated tap water, and 200 mL of the sludge inoculum were added to this vessel. The vessel was immediately placed in the temperature-controlled incubator and aerated.

REFERENCE SUBSTANCE FLASK
16 mL of the synthetic sewage feed, 274 mL of dechlorinated water, 10 mL of the reference toxicant stock solution and 200 mL of the sludge inoculum were added to the vessel. The vessel was immediately placed in the temperature-controlled incubator and aerated. This process was repeated at 15 minute intervals with 20 mL and 40 mL of reference substance toxicant solution respectively. For each reference toxicant addition, the volume of dechlorinated water was adjusted so that the total volume within the test vessel remained at 500 mL.

EFFECT PARAMETERS MEASUERD: ASSESSMENT OF OXYGEN CONSUMPTION
After three hours of exposure, or in the case of the controls, three hours within the test vessel, 300 mL of the sludge mixture were transferred into a BOD bottle containing a teflon-coated magnetic stir bar. The bottle was capped with the BOD bottle oxygen probe to eliminate air space and placed on a magnetic stirring plate. The sludge mixture was stirred at a constant rate and the consumption of oxygen over time was recorded on a strip chart recorder. The test material control or reference toxicant concentration was written on the strip chart paper. The oxygen readings were recorded for ten minutes or until a linear trace covering a sufficient range of oxygen concentrations was obtained.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 d
Dose descriptor:
EC50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 d
Dose descriptor:
other: NOAEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 d
Dose descriptor:
IC10
Effect conc.:
1 767 other: ppm
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95 % confidence intervals are 892.0 - 2642.0
Details on results:
An IC10 was calculated using a point estimate technique and Jackknifed to produce a variance from which a confidence could be derived. The result estimate for the ICI0 was 1767 ppm of the test material. The 95 % confidence interval for this estimate is from 892.0 to 2642.0 ppm.

- Blank controls oxygen uptake rate: The first control vessel showed a change of 4.2 mg/L for oxygen concentration for a 3 2/6-minute period. This equates to an oxygen consumption rate of 75.7 mg O2/L*hr. The second control vessel showed a change in oxygen concentration of 15 mg/L for 24/6 minutes. The second control thus had an oxygen consumption rate of 78.7 mg O2/L*hr. The average for the two controls was 77.2 mg O2/L*hr.
Results with reference substance (positive control):
Test inoculum was exposed to three concentrations of 3,5-dichlorophenol: 40, 20, and 10 ppm. The 40-ppm test concentration showed a change of 0.80 mg O2/L for 6 2/6 minutes, or a respiration rate of 7.6 mg O2/L*hr. The sludge exposed to 20 ppm of the reference toxicant produced an oxygen consumption of 4.5 mg O2/L over a 5 1/6-minute period with the respiration rate being equal to 52.2 mg O2/L*hr. The 10-ppm test inoculum consumed 4.5 mg O2/L over a period of 5 2/6 minutes with a respiration rate equal to 50.7 mg O2/L*hr.
The resultant consumption rates for the inoculum exposed to the reference toxicant expressed as a percentage of the control were used to calculate the EC50 of the reference toxicant in the sludge used to test the experimental material. Expressed as a percent inhibition compared to the control: The microbes in the 40-ppm test were 90.2 % inhibited: the microbes in the 20 ppm test were 32.4 % inhibited; and the microbes in the 10-ppm test were 34.3 % inhibited. Plotting percent effect (% inhibition) as a probit value and concentration as a log (base 10) value, the EC50 was calculated to be 18.5 mg 3,5-dichlorophenol per litre.
Reported statistics and error estimates:
The resultant respiration rates were respectively; 78.7, 69.1, 69.1, 89.8, and 36.0 mg O2/L*hr. These data translate into percent inhibitions of -1.9 %, 10.5 %, 10.5%, -16.3 %, and 53.4 %. Even though the 1 000 ppm concentration did produce an inhibition greater than 50 %, an EC50 could not be calculated. The best estimate for the EC50 is that it exceeds 10 000 mg/L. Each respiration rate was statistically compared as a single value versus the mean of the controls following the procedures of Sokal & Rohlf (1981) using a single-sided t-test. The results of the t-test revealed that the 10 000 mg/L test concentration expressed a statistically significant inhibition of oxygen consumption as compared to the controls. The discriminatory power of the test is low, however, due to having only one degree of freedom.

Table 1: Summary of Data

Control Data

Sample Number

Change in O2
(mg/L)

Change in Time
(minutes)

O2 Consumption
(mg/L*hr)

Sample # 1

4.2

3 2/6 

75.7

Sample # 2

3.5

 2 4/6

78.7

Reference Toxicant Data

Test Concentration
(mg/L)

Change in O2
(mg/L)

Change in Time
(minutes)

O2 Consumption
(mg/L*hr)

Percent Inhibition

10

4.5

 5 2/6

50.7

34.3

20

4.5

 5 1/6

52.2

32.4

40

0.8

 6 2/6

7.6

90.2

Reference Toxicant EC50 = 18.5 mg/L

Test Material Data

Test Concentration
(mg/L)

Change in O2
(mg/L)

Change in Time
(minutes)

O2 Consumption
(mg/L*hr)

Percent Inhibition

1

3.5

 2 4/6

78.7

-1.9

10

2.5

 2 1/6

69.1

10.5

100

2.5

 2 1/6

69.1

10.5

1000

2.5

 1 4/6

89.8

-16.3

10000

3.0

 5

36.0

53.4

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the EC50 of the test material was estimated to be greater than 10 000 mg/L.
Executive summary:

The potential impact of the test material on microbial metabolism, as represented by the consumption of oxygen, was investigated using the "Activated Sludge, Respiration Inhibition Test" as prescribed by OECD209 (Expanded Range Procedures) and performed under GLP conditions.

The test duration was a three-hour exposure period to the test material followed by up to ten minutes for the measurement of oxygen consumption. The study design was comprised of five nominal exposure concentrations: 1, 10, 100, 1 000 and 10 000 ppm; a duplicate control group; and an assessment of the sensitivity of the inoculum used in the test to a reference toxicant (3,5-dichlorophenol).

 The activated sludge respiration test with the test material passed the quality control criteria for an acceptable test. The EC50 calculated for the reference toxicant was 18.5 mg/L, within the acceptable range of 5 to 30 mg/L. The two control replicates produced oxygen consumption rates within the required 15 % of each other, 75.7 and 78.7 mg O2/L*hr.

 The respiration rates of the sludge-associated microbes exposed to the five nominal concentrations of the test material were 78.7, 69.1, 69.1, 89.8 and 36.0 mg O2/L*hr respectively. The calculated no-observed-adverse-effect concentration (NOAEC) based on inhibition compared to the control was calculated to be equal to 1000 ppm.

 Under the conditions of this study, the EC50 for the test material regarding the inhibition of metabolism, as represented by respiration, is estimated to be greater than 10 000 mg/L. The NOAEC was calculated to be equal to 1 000 mg/L. The IC10 was calculated to be 1767 ppm with a 95 % confidence interval from 892.0 to 2642.0 ppm.

The test is limited in that it represents a screening test of the potential effect of the test material on microbial metabolism.

Description of key information

The EC50 was > 10 000 mg/L and the NOAEC was 1 000 mg/L, based on inhibition.

Key value for chemical safety assessment

EC50 for microorganisms:
10 000 mg/L

Additional information

The potential impact of the test material on microbial metabolism, as represented by the consumption of oxygen, was investigated using the "Activated Sludge, Respiration Inhibition Test" as prescribed by OECD 209 (Expanded Range Procedures) and performed under GLP conditions.

The test duration was a three-hour exposure period to the test material followed by up to ten minutes for the measurement of oxygen consumption. The study design was comprised of five nominal exposure concentrations: 1, 10, 100, 1 000 and 10 000 ppm; a duplicate control group; and an assessment of the sensitivity of the inoculum used in the test to a reference toxicant (3,5-dichlorophenol).

 The activated sludge respiration test with the test material passed the quality control criteria for an acceptable test. The EC50 calculated for the reference toxicant was 18.5 mg/L, within the acceptable range of 5 to 30 mg/L. The two control replicates produced oxygen consumption rates within the required 15 % of each other, 75.7 and 78.7 mg O2/L*hr.

 The respiration rates of the sludge-associated microbes exposed to the five nominal concentrations of the test material were 78.7, 69.1, 69.1, 89.8 and 36.0 mg O2/L*hr respectively. The calculated no-observed-adverse-effect concentration (NOAEC) based on inhibition compared to the control was calculated to be equal to 1000 ppm.

 Under the conditions of this study, the EC50 for the test material regarding the inhibition of metabolism, as represented by respiration, is estimated to be greater than 10 000 mg/L. The NOAEC was calculated to be equal to 1 000 mg/L. The IC10 was calculated to be 1767 ppm with a 95 % confidence interval from 892.0 to 2642.0 ppm.

The test is limited in that it represents a screening test of the potential effect of the test material on microbial metabolism.