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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
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
GLP compliance:
yes
Specific details on test material used for the study:
- Purity: 100%.
- Composition of test material: Composition meets ISO Standard 4730 Oil of Melaleuca, terpinen-4-ol type (Tea Tree Oil).
- Lot (batch) No.: 1215
- Lab No.: 1536
- Expiration date of the lot: 30 March 2009
- Storage condition of test material: Room temperature in the dark.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The results of a formulation trial showed that the test substance floated on the surface of dechlorinated water at the highest concentration employed in the first test (1000 mg/l) and was considered potentially volatile. Therefore, in order to minimise loss of the material by air stripping from air sparged cultures, the tests were conducted using two litre Erlenmeyer flasks and the contents aerated on an orbital shaker for thirty minutes at a constant speed of ca. 200 rpm. This deviation from guideline is considered to be appropriate and would not have adversely affected the outcome of the study.

Test concentrations were established by the addition of appropriate volumes of the test substance added using an automatic pipette to flasks containing 284 ml of dechlorinated water immediately before test set up. Additions of synthetic sewage and inoculum were then made at fifteen minute intervals to give a final volume of 500 ml according to the schedule outlined in Table 1 (see 'Any other information on materials and methods').

Preparation of solutions of the reference substance: A concentrated solution of 3,5-DCP (500 mg/l) was prepared by dissolving 0.5 g in 10 ml of 1N sodium hydroxide and diluting to approximately 30 ml with ultrapure water. Sulphuric acid (1N) was added to the point of incipient precipitation and the solution made up to a final volume of one litre with ultrapure water. The pH of this solution was then measured. Nominal concentrations of 3, 10 and 32 mg/l were prepared by dilution of this concentrated solution.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Worlingworth Sewage Treatment Works
- Method of cultivation: A sample of activated sludge was obtained the day before the start of the tests. In the laboratory, the samples were maintained under aerobic conditions until required.
- Preparation of inoculum for exposure: The concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the tests. On the day of collection, aliquots of the activated sludge were filtered through dried and preweighed Whatman GF/C filter papers which were then dried again at approximately 105°C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage was added to each stock of activated sludge and these were aerated overnight. On the day of the test, the MLSS content of the sludge was determined and adjusted to 4 g/l by the addition of dechlorinated tap water. The pH of the sludge was also measured.

- Pretreatment: 50 ml of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. Synthetic sewage feed contains 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H20, 0.2 g MgSO4.7H20 and 2.8 g K2HPO4. Dissolved in ultrapure water and made up to 1 litre.
- Initial cell concentration: Number of micro-organisms was determined as the MLSS content of the test medium. The content was adjusted to 4 g/l for the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Post exposure observation period:
Following the exposure period, a well-mixed sample of each mixture was transferred to a biochemical oxygen demand (BOD) bottle (nominal capacity, 270 ml). The rate of oxygen consumption was measured over a period of approximately 10 minutes or until the dissolved oxygen concentration fell below 2 mgO2/l using a Yellow Springs Instrument (YSI) dissolved oxygen meter, with temperature probe and self stirring bottle probe, connected to a chart recorder.
Test temperature:
The temperature of the samples was measured at the start and end of the test. The measurements are given in Tables 3 and 5 respectively (see 'Any other information on material and methods' below).
pH:
The pH of the samples was measured at the start and end of the test. The measurements are given in Tables 3 and 5 respectively (see 'Any other information on material and methods' below). Measurements of the pH of aqueous stock solutions of the reference substance and of samples of activated sludge (4 g/l) before the start of the tests are as follows:
3,5-DCP stock solution: 7.9 for preliminary test; 7.9 for definitive test
Activated sludge: 7.5 for preliminary test; 7.4 for definitive test
Dissolved oxygen:
Dissolved oxygen concentrations and measurement times are given in Tables 2 and 4 for the preliminary and definitive tests, respectively.
Nominal and measured concentrations:
Nominal concentrations of test substance: 75, 150, 300, 600, 1200 mg/l
Details on test conditions:
- Culturing apparatus: Two-litre Erlenmayer flasks.
- Number of culture flasks/concentration: One culture flask was used at each concentration.
- Preparation of synthetic sewage: Synthetic sewage feed for activated sludge was prepared by dissolving the following in one litre of ultrapure water: peptone (16.0 g), meat extract (11.0 g), urea (3.0 g), sodium chloride (0.7 g), calcium chloride dihydrate (0.4 g), magnesium sulphate heptahydrate (0.2 g), di-potassium hydrogen phosphate (2.8 g).
- Dilution water: The dilution water used to prepare solutions of synthetic sewage and the reference substance was tap water that had been softened and treated by reverse osmosis (Prima 4 reverse osmosis unit) and then purified (UHP unit) to give a nominal resistivity ≥18 MegOhm.cm. The water used to prepare the test mixtures was dechlorinated tap water (hardness 200 - 250 mg/l as CaCO3).

- Preliminary test concentrations: 10, 100, 1000 mg/l
- Effect data: Significant inhibition of respiration of the sludge was recorded (see Table 3 in 'Any other information on materials and methods' below).
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (3,5-DCP), 99.9% (employed in both tests at 3, 10 and 32 mg/l).
Duration:
30 min
Dose descriptor:
other: EC20
Effect conc.:
77.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% confidence limits: 48.1 - 114 mg/l
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
257 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% confidence limits: 203 -325 mg/l
Duration:
30 min
Dose descriptor:
other: EC80
Effect conc.:
859 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% confidence limits: 632 - 1250 mg/l
Details on results:
Figure 1 illustrates the effect of the test substance on the respiration rate of the sludge in the definitive test.
The specific respiration rates of control cultures established at the end of the two test series (24.2 and 21.4 mgO2/g/h) were 90% and 110% of the rates of those established at the start (26.8 and 19.5 mgO2/g/h). These results show that the tests were valid and that the sample of activated sludge employed was sensitive to inhibition.
Concentration-related inhibition of respiration rates was seen at each tea tree oil concentration (see Table 5 in 'Any other information on materials and methods' for values). The highest tested concentration of tea tree oil in the definitive test (1200 mg/l) caused 86% inhibition. The EC50 of the test substance was calculated from the results of the definitive test to be 257 mg/l (95% confidence limits, 203 -325 mg/l). The EC20 and EC80 values were calculated to be 77.1 and 859 mg/l, with 95% confidence limits of 48.1 - 114 and 632 - 1250 mg/l, respectively.
Results with reference substance (positive control):
Figure 1 illustrates the effect of 3,5-DCP on the respiration rate of the sludge in the definitive test. Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP. The thirty-minute 50% effect concentration (EC50) for 3,5-DCP was calculated to be 9.8 mg/l (95% confidence limits, 7.1 - 13.6 mg/l) in the preliminary test, and 8.2 mg/l (95% confidence limits, 6.3 - 10.7 mg/l) in the definitive test. These results show that the tests were valid and that the sample of activated sludge employed was sensitive to inhibition.
Reported statistics and error estimates:
The EC50 and 95% confidence limits (Donaldson and Schnabel, 1985) of the test and reference substances were calculated using the SAFEstat curvefit programme (SAS Institute 1999). Since the permitted maximum difference for specific respiration rates in control beakers established at the beginning and the end of the test period was 15%, this value was used to define the criteria for biologically significant levels of inhibition in mixtures containing the test substance.

Calculation of results:

The oxygen consumption rate of each test, reference and control mixture was calculated from oxygen levels in the following way: r = (DO(1) - DO(2)) / t

where:

r = oxygen consumption rate (mgO2/l/minute)

DO(1) = initial oxygen level (mgO2/l)

DO(2) = final oxygen level (mgO2/l)

t = time over which measurements were made (minutes)

The specific respiration rate of each mixture was calculated from the rate of oxygen consumption in the following way: SRR = (r x 60)/MLSS

where:

SRR = specific respiration rate (mgO2/g/h)

MLSS = concentration of mixed liquor suspended solids in the sample of activated sludge (g/l) in the test or control mixture.

The inhibitory effect of the test or reference substance at a particular concentration was calculated by expressing the specific respiration rate as a percentage of the mean of the respiration rates of the two controls in the following way: % inhibition = 1 - ((2 Rs) / (Rc1 + Rc2)) x 100

where:

Rs = rate of oxygen consumption of test or reference substance

Rc1 = rate of oxygen consumption of control 1

Rc2 = rate of oxygen consumption of control 2

Validity criteria fulfilled:
yes
Conclusions:
Concentration-related inhibition of respiration rates occurred at each tea tree oil concentration in the definitive test. The highest tested concentration of tea tree oil in the definitive test (1200 mg/l) caused 86% inhibition. The EC50 of the test substance was calculated from the results of the definitive test to be 257 mg/l (95% confidence limits, 203 -325 mg/l). The EC20 and EC80 values were calculated to be 77.1 and 859 mg/l, with 95% confidence limits of 48.1 - 114 and 632 - 1250 mg/l, respectively. The thirty-minute EC50 for 3,5-DCP (9.8 mg/l in the preliminary test and 8.2 mg/l in the definitive test) fulfilled the validity criterion relating to sensitivity to inhibition (acceptable EC50 range 5 to 30 mg/l), and that relating to the respiration rates in the control (variation not greater than 15%) was also satisfied.
Executive summary:

The effect of tea tree oil on the respiration rate of activated sludge was assessed by the methods detailed in OECD Test Guideline 209.  Samples of activated sludge (suspended solids 1.6 g/l) fed with synthetic sewage were exposed for thirty minutes to dilutions of the test substance.  Their rates of oxygen consumption were determined using an oxygen electrode and compared with those of the controls.  Tea tree oil was employed at nominal concentrations of 75, 150, 300, 600 and 1200 mg/l.  The reference inhibitor 3,5-dichlorophenol (3,5-DCP) was employed at 3, 10 and 32 mg/l, as a positive control.  The specific respiration rate of the control culture established at the end of the test series was 110% of the rate of that established at the start.  The thirty-minute 50% effect concentration (EC50) for 3,5-DCP in the definitive test was calculated to be 8.2 mg/l.  These results show that the tests were valid and that the samples of activated sludge employed were sensitive to inhibition.  The EC50 of the test substance was calculated from the results of the definitive test to be 257 mg/l.

Description of key information

Key value for chemical safety assessment

EC50 for microorganisms:
257 mg/L

Additional information