Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD and EU method and under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
Deviation was considered to have had no adverse effect on the study
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Boronic Acid
Chemical Name: 6-hydroxy-2-naphtaleneboronic acid
- Substance type: off white powder
- Expiration date of the lot/batch: 13 June 2014
- Storage condition of test material: initially room temperatire in the dark for 7 days, thereafter approximately 4°C in the dark
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Reference Item
Information as provided by the supplier (Sigma-Aldrich).

Identification: 3,5-Dichlorophenol
Description: pale brown crystalline solid
Batch: MKBK4755V
Purity: 97%
Expiry Date: 26 September 2014
Storage Conditions: room temperature

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
- Concentrations: Based on the results of a range-finding test the following test concentrations were assigned to the definitive test in order to obtain an EC50 value for the test item: 5.6, 18, 56, 180 and 560 mg/L.
- Sampling method:
- Sample storage conditions before analysis: The test was conducted under normal laboratory lighting in a temperature controlled room at approximately 20 °C.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
From the range-finding test and subsequent extra solubility work, the test item was found to be
soluble in water up to 500 mg/L. Therefore, the test item was dissolved directly in water to obtain
the test concentrations of 5.6, 18, 56 and 180 mg/L and dispersed directly in water for the test
concentration of 560 mg/L.

An amount of test item (500 mg) was dissolved in water and the volume adjusted to 1 liter to give a
500 mg/L stock solution from which a dilution was made to give a 50 mg/L stock solution. An aliquot
(56 mL) of the 50 mg/L stock solution was washed into a 500 mL conical flask and the volume
adjusted to 200 mL with water. Similarly, aliquots (18, 56 and 180 mL) of the 500 mg/L stock
solution were washed into separate 500 mL conical flasks and the volume adjusted to 200 mL with
water. In order to maintain consistency with the 560 mg/L test vessels, these vessels were subjected
to magnetic stirring for 24 hours. 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 5.6, 18, 56, and 180 mg/L (3 replicates of each).

The volumetric flasks containing the stock solutions were inverted several times to ensure
homogeneity of the stock solutions.

An amount of test item (280 mg (in triplicate)) was 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 concentration of 560 mg/L (3 replicates).

The pH of the test item solutions/dispersions was measured after stirring using a WTW pH330 pH
meter and adjusted to between pH 7.0 to pH 8.0.

The control group was maintained under identical conditions but not exposed to the test item.

Reference Item Preparation:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and was used on the day of collection. The pH of the sample was 7.6 measured using a WTW pH 330 pH meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GFIA 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 °C 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.


Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and was used on the day of collection. The pH of the sample was 7.6 measured using a WTW pH 330 pH meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GFIA 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 °C 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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Observations were made on the test preparations throughout the test period. Observations of the test item vessels at O hours were made prior to addition of activated sewage sludge.

Test conditions

Hardness:
not applicable
Test temperature:
approximately 20°C througout testing
pH:
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 3401 pH and dissolved oxygen meter.
Dissolved oxygen:
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 3401 pH and dissolved oxygen meter.
Salinity:
not applicable
Nominal and measured concentrations:
Range finding test- nominal test concentrations of 10, 100 and 1000mg/L. Definitive test- test concentrations: 5.6, 18, 56, 180 and 560 mg/L.

Details on test conditions:
TEST SYSTEM
- Test vessel: conical flask
- No. of vessels per concentration (replicates): 3 replicates of each
- No. of vessels per control (replicates): 3
-Aeration: The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1.0 liter per minute


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized reverse osmosis water
- Total organic carbon: containing less than 1mg/L Dissolved Organic Carbon (DOC)


OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test item solutions/dispersions was measured after stirring using a WTW pH330 pH meter and adjusted to between pH 7.0 to pH 8.0.
- Photoperiod: All test vessels were shielded from light during mixing


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-pH Measurements
-Oxygen Concentrations
-Measuremenat of the Respiration Rates

TEST CONCENTRATIONS
- Spacing factor for test concentrations:

- Range finding study: activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dispersed directly in water. 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 concentrations: Based on the results of the range-finding test the following test concentrations were assigned to the definitive test in order to obtain an EC50 value for the test item: 5.6, 18, 56, 180 and 560 mg/L.

- Results used to determine the conditions for the definitive study: From the range-finding test and subsequent extra solubility work, the test item was found to be soluble in water up to 500 mg/L. Therefore, the test item was dissolved directly in water to obtain the test concentrations of 5.6, 18, 56 and 180 mg/L and dispersed directly in water for the test concentration of 560 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
92 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 limits 70-120 mg/L
Results with reference substance (positive control):
- Results with reference substance valid? Yes

Reported statistics and error estimates:
In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7.0 mg 02/L and 2.0 mg 02/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.

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg 02/L with the exception of control replicates 3 and 4 showing 53% to 55% saturation. This deviation was considered to have had no adverse effect on the study as all of the control vessels showed a similar oxygen consumption value indicating that an initial low oxygen content had no effect on the final result.

Any other information on results incl. tables

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 02/L and 2.0 mg 02/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


Applicant's summary and conclusion

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 92 mg/L, 95% confidence limits 70- 121 mg/L.

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.
Executive summary:

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge.  The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous solution or dispersion of the test item at concentrations of 5.6, 18, 56, 180 and 560 mg/L (3 replicates) for a period of 3 hours at a temperature of approximately 20 °C with the addition of a synthetic sewage as a respiratory substrate.

 

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50

value of 92 mg/L, 95% confidence limits 70- 120 mg/L.

The reference item gave a 3-Hour EC50 value of 7.5 mg/L, 95% confidence limits 5.9 - 9.6 mg/L.