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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 July - 19 July 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
not specified
GLP compliance:
yes
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:
No data.
Vehicle:
no
Details on test solutions:
Each time that a subsample was removed from the sample container, the entire sample was placed in a heated water bath adjusted to approximately 60°C for approximately 30 minutes to liquefy the test substance.

DILUTION WATER.
Water used for dilution of the sludge and for all toxicity testing was deionized water collected at Marblehead, Massachusetts with a hardness of 20 mg/L as CaC03.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge used for the final range-finding and definitive tests was obtained from the municipal wastewater treatment plant at Newburyport, Massachusetts on July 16, 1996. This treatment plant treats predominantly domestic waste.

As collected, the suspended solids concentration of the sludge was 5.8 g/L. It had a suspended solids content of 3.8 g/L on the day of use for the definitive test. Because the sludge was not used the day it was collected, synthetic sewage, consisting of 16 g/L peptone, 11 g/L, meat extract, 3 g/L urea, 0.7 g/L NaCl, 0.4 g/L CaC12.H20, 0.2 g/L MgS04.7H20, and 2.8 g/L K2HP04 was combined with the sludge at the rate of approximately 50 ml/L. The sludge was incubated at room temperature and aerated prior to use. The sludge was adjusted to pH 8.0 with 0.1N hydrochloric acid at the time of use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
No data.
Hardness:
hardness of 20 mg/L as CaC03
Test temperature:
20.7 - 20.8C
pH:
pH sludge adjusted to 8 with NaOH
Dissolved oxygen:
See Table 1
Salinity:
No data.
Nominal and measured concentrations:
Nominal concentrations of test substance were: 0 (control), 1.0, 3.2, 10, 32, and 100 mg/L.
Details on test conditions:
A range-finding test was attempted on July 11, 1996, but results were inconclusive because respiration rates of the duplicate controls differed from each other by more than 15%. A second range-finding test was performed on July 18, 1996 with a control and 5 concentrations of RH-573 Technical (5.0, 10, 50, 100, and 500 m a ) . Respiration rates at the 2 lowest concentrations of RH-573 Technical were similar to the control rate, and the respiration rates at 50, 100, and 500 mg/L were 15, 5, and 0% of the control, respectively.

The definitive respiration inhibition test was conducted for 3 hours on July 19, 1996. Nominal concentrations of test substance were: 0 (control), 1.0, 3.2, 10, 32, and 100 mg/L. A 200 mg/L stock solution was prepared by combining 0.256 g test substance and deionized water and adjusting the final water volume to 500 mL in a Class A volumetric flask. Concentrations were formulated by the addition of stock solution to dilution water without the use of a solvent.

The test was conducted under static conditions at 20 +/- 2C. At time 0 for each test vessel, 16 mL of synthetic sewage was brought up to 300 mL in dilution water containing the appropriate concentration of test substance. To this mixture, which was placed in a 1 liter glass beaker, was added 200 mL of microbial inoculum. Test vessels were established at 10 minute intervals and vessels were aerated with 0.5 to 1.0 liters of air per minute for 3 hours. The first and last vessels established were controls.

After the 3 hour incubation period the contents of each test vessel were transferred to a BOD bottle equipped with a magnetic stirrer and the dissolved oxygen concentration in this sealed bottle was monitored for 10 minutes. Test vessels were maintained under ambient laboratory lighting during the test (light intensity = 8 uEin/m2sec). Dissolved oxygen was measured with a YSI Model 57 meter (instrument number 18).

A respiration inhibition test was performed on July 18, 1996 with the batch of sludge used for this definitive test and the reference toxicant 3,s-dichlorophenol. The test was performed with 3 nominal concentrations of 3,5-dichlorophenol (5.0, 12, and 30 mg/L) under test conditions described above
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% confidence interval of 3.2 to 100 mg/L.
Details on results:
Insoluble test substance was not noted during the test. Data generated by the respiration inhibition test are presented in Table 1. The control respiration rates were within 15% of each other and the temperature during the test was 20.7 to 20.8°C.

The test substance inhibited respiration of the activated sludge in the tested range of concentrations. The 3 hour EC50 for activated sludge exposed to RH-573 Technical was 41 mg/L with a 95% confidence interval of 3.2 to 100 mg/L.
Results with reference substance (positive control):
The
EC50 determined during a reference toxicant test with the batch of activated sludge used for the definitive test and 3,5-dichlorophenol was 16 mg/L and within the acceptable range of 5 to 30 mg/L.
Reported statistics and error estimates:
The 3 hour EC50 for activated sludge exposed to RH-573 Technical was 41 mg/L with a 95% confidence interval of 3.2 to 100 mg/L.

Table 1. Activated Sludge Respiration Data from the Test with RH-573 Technical.

 Nominal Concentration       Oxygen Concentration (mg/L)  Time to 6.5 mg/L  Time to 2.5 mg/L  Respiration Ratec
 of RH-573 Technical (mg/L)  Rep.  Initial  Final  Oxygen (min)  Oxygen (min)  (mg/L/hr)
 0 (control)  1  6.5  0.1  --a  2.0  120
   2  6.2  0.1  --  2.0  111
 1.0  1  5.6  0.1  --  2.0  93
   2  5.6  0.1  --  3.0  62
 3.2  1  5.5  0.1  --  2.0  90
   2  5.6  0.1  --  2.0  93
 10  1  6.2  0.1  --  3.0  74
   2  5.6  0.1  --  3.0  62
 32  1  4.9  0.1  --  2.0  72
   2  4.9  0.1  --  2.0  72
 100  1  7.8  6.0  8.0  --b  11
   2  7.8  5.9  7.0  --  11

aDashes in this column indicate that an initial dissolved oxygen concentration was less than 6.5 mg/L.

bDashes in this column indicate that a dissolved oxygen concentration did not reach 2.5 mg/L.

cThe test substance inhibited respiration of the activated sludge in the tested range of concentrations. The 3 hour EC50 for activated sludge exposed to RH-573 Technical was 41 mg/L with a 95% confidence interval of 3.2 to 100 mg/L. The EC50 was calculated using the binornial/nonlinear interpolation method.

Validity criteria fulfilled:
yes
Conclusions:
The test substance inhibited respiration of the activated sludge in the tested range of concentrations. Exposure of activated sludge to RH-573 Technical resulted in a 3 hour EC50 of 41 mg/L with a 95% confidence interval of 3.2 to 100 mg/L.
Executive summary:

The inhibition of activated sludge respiration by RH-573 Technical (Lot No. SW 1029, 97.8% a.i., TD No. 95-100) was investigated during a study conducted at T.R. Wilbury Laboratories. The test, which was designed to determine the 3 hour median effective concentration (EC50) of the test substance using activated sludge, was performed on July 19, 1996 according to Rohm and Haas Protocol No. 95P- 165 and T.R. Wilbury No. 103 7-RH.

The test substance was supplied by the sponsor and delivered to the laboratory on June 6, 1996. The test was performed at 20 ± 2°C under static conditions with a control and five concentrations of test substance (1.0, 3.2, 10, 32, and 100 mg/L). Nominal concentrations of RH-573 Technical were used for all calculations. No insoluble material was noted during the test.

Activated sludge was obtained from the municipal wastewater treatment plant in Newburyport, Massachusetts on July 16, 1996. The total suspended solids content of the activated sludge was 5.8 g/L on the day of collection and 3.8 g/L on the day of the respiration inhibition test. The EC50 determined during a reference toxicant test with this batch of activated sludge and 3, 5- dichlorophenol was 16 mg/L and within the acceptable range of 5 to 30 mg/L.

The test substance inhibited respiration of the activated sludge in the tested range of concentrations. Exposure of activated sludge to RH-573 Technical resulted in a 3 hour EC50 of 41 mg/L with a 95% confidence interval of 3.2 to 100 mg/L.

Description of key information

The inhibition of activated sludge respiration by RH-573 Technical (97.8% a.i.) was investigated. The test substance inhibited respiration of the activated sludge in the tested range of concentrations.

Exposure of activated sludge to RH-573 Technical resulted in a 3 hour EC50 of 41 mg/L with a 95% confidence interval of 3.2 to 100 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
41 mg/L

Additional information