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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:
03 to 04 September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, conducted to current OECD Guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
The dissolved oxygen content was not above 60% to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L in several of the vessels.This deviation was considered to have no adverse effect on the study as all oxygen consumption values were measured over the linear portion of the trace.
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:
yes
Details on sampling:
As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 7.0 mg O2/L and 2.0 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.
Vehicle:
no
Details on test solutions:
Amounts of test item (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 03 September 2013 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:16 g Peptone11 g Meat extract3 g Urea0.7 g NaCl0.4 g CaCl2.2H2O0.2 g MgSO4.7H2O2.8 g K2HPO4dissolved in 1 liter of water with the aid of ultrasonication.The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (5 liters) was fed synthetic sewage sludge (250 mL). The pH of the sample on the day of the test was 7.8 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Determination of the suspended solids level of the activated sewage sludge on the day of the test was carried out by filtering a sample (50 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
Not measured
Test temperature:
approximately 20 °C
pH:
The pH of the test item dispersions was measured after stirring using a WTW pH/Oxi 340I pH and dissolved oxygen meter (see Table 1) and adjusted to between pH 7.0 and 8.0 if necessary.
Dissolved oxygen:
The oxygen concentrations in all vessels were measured after 30 minutes contact time. Refer to Table 2 in attachments below.
Salinity:
Not applicable - freshwater
Nominal and measured concentrations:
10, 100 and 1000 mg/L (3 replicates).
Details on test conditions:
Observations Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge. pH Measurements The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Oxygen Concentration The oxygen concentrations in all vessels were measured after 30 minutes contact time (see Table 2). Measurement of the Respiration Rates As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 7.0 mg O2/L and 2.0 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period. Calculation of the Oxygen Uptake Rates The respiration rate, R, expressed in milligrams oxygen per liter per hour (mg O2/L/h), was calculated from the linear part of the recorded oxygen decrease graph according to the following equation: R = [(Q1 - Q2) / dt] x 60 Where:Q1 = the oxygen concentration at the beginning of the selected section of the linear phase (mg/L); Q2 = the oxygen concentration at the end of the selected section of the linear phase (mg/L); dt = the time interval between the beginning and end of the selected section of the linear phase (min). The specific respiration rate, RS, expressed as the amount of oxygen consumed per gram dry weight of sludge per hour (mg O2/g/h) was deduced according to the following equation: RS = R / SS Where: SS = the concentration of suspended solids in the test mixture (g/L). Calculation of Percentage of InhibitionThe percentage inhibition was calculated according to the following equation: % inhibition = [1 – (R/Rbc)] x 100 Where: Rbc = the mean respiration rate of the blank controls.ECX and NOEC The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line. 95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949). One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data for the range-finding test after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).Validity Criteria The results of the study are considered valid if (i) the EC50 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 2 to 25 mg/L (ii) the specific respiration rate of the blank controls is not less than 20 mg oxygen per gram dry weight of sludge per hour (iii) the coefficient of variation of oxygen uptake rate in control replicates is not more than 30% at the end of the test.
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.
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 dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 2 (attached). Oxygen consumption rates and percentage inhibition values for the control, test and reference items are given in Table 3 (attached). The pH values of the test preparations at the start and end of the exposure period are given in Table 4 (attached). Observations made on the test preparations throughout the range-finding test are given in Table 5 (attached). The dissolved oxygen concentrations after 30 minutes contact time in several of the vessels were below 60% of the dissolved oxygen saturation level of 8.9 mg O2/L. This deviation to the study plan was considered to have had no adverse effect on the study given that all oxygen consumption values were measured/calculated over the linear portion of the traces. No statistically significant toxic effects were shown at all of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The coefficient of variation of oxygen uptake in the control vessels was 2.9% and the specific respiration rate of the controls was 21.97 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied. In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7.0 mg O2/L and 2.0 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.
Results with reference substance (positive control):
The reference item gave a 3-Hour EC50 value of 12 mg/L.

The following results were derived:

 

NovaSpec 450

3,5-dichlorophenol

 

ECx (3 Hours) (mg/L)

ECx (3 Hours) (mg/L)

C10

-

2.8

EC20

-

4.0

EC50

>1000

12

EC80

-

36

NOEC

1000

-

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. The substance is not considered to be inhibitory to sewage sludge microorganisms. No classification is necessary.
Executive summary:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. The substance is not considered to be inhibitory to sewage sludge microorganisms. No classification is necessary.

Description of key information

Assessment of toxicity to microorganisms

Key value for chemical safety assessment

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

Additional information

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. The substance is not considered to be inhibitory to sewage sludge microorganisms. No classification is necessary.