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Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms, other
Remarks:
based on data from a OECD TG 301F study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
(version 1992)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
(See "details on test conditions")
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: Mixed liquor (activated sludge) was obtained from an aeration tank containing Mixed Liquor Suspended Solids (MLSS) at the University of British Columbia domestic wastewater treatment pilot plant. Approximately 3 L of mixed liquor (activated sludge) was obtained on 8 June 2005 at 11:55 AM for use in the test.
- Date of receipt: June 8, 2005
- Volume received: Approximately 3 L
- Inoculum pre-test treatment: Sludge was centrifuged, supernatant discarded, and sludge pellet re-suspended in mineral media twice; sludge was brought up to the original volume; aerated in the dark at ambient temperature for 1 day before use
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
Test temperature:
24 ± 0.6°C
pH:
7.6 at test initiation
7.4 - 7.8 at test completion
Nominal and measured concentrations:
- Nominal concentration: 35 mg/L
- Measured concentration: no analysis performed (or required)
Details on test conditions:
STUDY DESIGN (SUMMARY)
- Inoculum concentration in test vessels: 19.8 mg/L (concentration of suspended solids)
- Photoperiod: In the dark (bottles were wrapped in aluminium foil)
- Test vessel: 2 L glass bottles, connected to a manometric respirometer and a controlled inlet source of ultra high purity grade oxygen
- Test volume: 2 L of each test solution was prepared; 0.5 L was removed; final test volume was 1.5 L
- Replicates of bottles: Two (Test and Inoculum Blank); One (Reference, Abiotic Control, and Toxicity Control)
- Controls: Inoculum blank; Reference substance (positive control); Toxicity controls (reference and test substance); Abiotic control
- Sampling days: Manometric respirometers were checked daily, and oxygen added to the test vessels only if required
- Reference substance concentration: 60 mg/L
- Test substance application: Test substance was weighed out and added directly to each appropriate test vessel

PREPARATION OF INOCULUM
The activated sludge was dispensed among ten 500 mL centrifuge bottles (ca. 3/4 full) and centrifuged at 3000 rpm for 10 min using a Beckman Coulter Model J2-21 centrifuge. The supernatant was discarded and the solid pellet re-suspended in mineral media and was centrifuged again at 3000 rpm. Then the pellet was re-suspended in mineral media to a final volume of ca. 3 L. The washed inoculum was aerated, stirring, at room temperature in the dark until use. The total pre-conditioning time prior to test initiation was 1 d.

PREPARATION OF MINERAL MEDIA
The mineral media was prepared on 6 June 2005 by adding the 400 mL of Solution A (8.5 g/L KH2PO4; 21.75 g/L K2HPO4; 33.4 g/L Na2HPO4·2H2O; 0.5 g/L NH4Cl) and 40 mL of Solutions B (36.4 g/L CaCl2·2H2O), C (22.5 g/L MgSO4·7H2O) and D (0.25 g/L FeCl3·6H2O) to Type I deionised water and brought to a final volume of 40 L. Stock solutions A to D were previously prepared on 21 April 2005 (Stock A), 2 February 2005 (Stocks B and C), and 21 April 2005 (Stock D), by dissolving each of the compounds in Type I deionised water to a total volume of 1 L. The pH of stock solution A was 7.4, which was within the required pH range (7.4 ± 0.2) for this solution. Mineral medium stock solution D was preserved by adding one drop of concentrated HCl. All chemicals were weighed using analytical balances, which were operated according to the procedures outlined in the Vizon SOP “Balance Operation”. All pH meters were operated according to the procedures outlined in the Vizon SOP “Operation/Calibration of the Fisher Scientific Accumet Basic pH Meter”.

PREPARATION OF STOCK SOLUTIONS
The published test guidelines (OECD 1992) suggest preparing a stock solution of the test solution, and preparing an initial batch solution to divide among the test vessels. Because of the low solubility of the test substance, this procedure was not feasible. Instead, the test substance was added directly to the test bottles. For each test bottle that was to contain test substance, an aliquot of 0.07 g of the test substance was weighed onto a watch glass using an analytical balance. The exact weight of the aliquot was recorded for each bottle, and the overall mean weight (± SD) was 0.0700 ± 0.0001 g. The aliquots were carefully rinsed from the watch glass into the appropriate test vessel using mineral media. The final nominal concentration of test substance in the test vessels was 35 mg/L.
A stock solution of 500 mg/L reference substance was prepared by adding 0.4998 g of reference substance to ca. 800 to 900 mL of mineral media to 1 L volumetric flask. The pH was measured and determined to be 7.6; therefore, the solution was made up to 1 L volume without any pH adjustment. A stock solution of 1000 mg/L sterilising agent was prepared by adding 0.0998 g of mercuric chloride to 100 mL of mineral media.

PREPARATION OF TEST SOLUTIONS
A series of five test solutions were used in the test: inoculum blank, test substance, reference substance, toxicity control and abiotic control. The inoculum blank and test substance were tested in duplicate.
The inoculum blank determined the biological oxygen demand in the absence of either the reference or the test substances. The reference substance is classified as readily biodegradable and was used as a positive control to assess test validity. The toxicity control bottle contained both test and reference substances and was used to determine if the test substance inhibited inoculum metabolism. The abiotic control was used to determine whether the test substance under went abiotic degradation during the test.
Test vessels were 2 L glass bottles connected to a manometric respirometer and a controlled inlet source of ultra high purity grade oxygen. The test solutions were prepared in the bottles by adding ca. 800 mL of mineral media to the bottles, and then adding the test substance to the appropriate bottles. Because of the low solubility of the test substance, the bottles were allowed to stir vigorously using a magnetic stir bar on magnetic stir plates for 1 hr in an attempt to create a dispersion. Reference substance, sterilising agent, and activated sludge were added to the appropriate bottles, and mineral media added to a total volume of 2 L. The test solutions were stirring during these additions to allow mixing. The bottles were removed from the stir plate in order to siphon 500 mL from each bottle which was used for initial pH measurements.
A CO2 scrubber was added to each bottle; the CO2 scrubber consisted of a small plastic vial that was filled with 3.99 ± 0.02 g (mean ± SD) of potassium hydroxide pellets and that was suspended from the underside of the bottle lid using stainless-steel wire. The bottles were attached to the manometric respirometers and oxygen inlets via a screw cap and rubber stopper and replaced on the magnetic stir plates.
The test solutions were stirred at ~400 rpm to ensure homogenous test solutions and to provide adequate oxygen exchange to create an aerobic environment in the test solutions. The bottles were also covered with aluminium foil to limit exposure to light during the daily readings. The test system was checked for air leaks, and then oxygen was added. The Study Plan stated that the test would be conducted at a temperature between 22 and 25°C, constant to within ± 1°C. The minimum and maximum room temperature during the exposure period was 22 and 25°C, although the mean room temperature was 24 ± 0.6°C.
Activated sludge (4 mL) was added to the inoculum blanks, test substance bottles, reference bottles, and toxicity controls. Activated sludge was not added to the abiotic controls. For the reference substance and toxicity control bottles, 240 mL of the 500 mg/L reference substance stock solution was added. For the abiotic control, 100 mL of the 1000 mg/L sterilising agent stock solution was added.

TEST OBSERVATIONS AND MEASUREMENTS
The test system was checked daily to ensure that the test solutions were stirring.
Readings of the manometric respirometers were conducted at least once daily. Readings were recorded separately for each column, and were later compiled to give pressure readings for each bottle in mm H2O. If necessary, pure oxygen was added to the bottles to maintain reasonable (readable) levels in the manometer columns. Ideally, after addition of oxygen, the left column would have a slightly higher positive reading than the right column. This setting allowed for the decrease in the internal (bottle) pressure due to oxygen consumption, while still maintaining readable levels on the columns. When the levels of the left column of the manometer were greater than 200; the value was estimated using a 155 mm ruler (1 mm = 1 mm H2O) because the manometers only have markings to 200. The high positive value of the left column indicated that the external (atmospheric) pressure was less than the internal pressure of the bottles. Changes in atmospheric pressure during the exposure period were compensated for by using the inoculum blanks to correct oxygen consumption in the test bottles. On Day 28, samples were taken from the bottles for analysis of pH.
Reference substance (positive control):
yes
Remarks:
benzoic acid, sodium salt / CAS No.: 532-32-1
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: ready biodegradability
Details on results:
See 'Any other information on results incl. tables'
Results with reference substance (positive control):
The biodegradation of the reference compound was 79% on Day 14.

Table: Nominal Concentration of Bottle Contents

Bottle

Inoculum (mg/L)

Test substance (mg/L)

Reference substance (mg/L)

Sterilizing agent (mg/L)

Test Substance Replicate #1

19.8

35

0

0

Test Substance Replicate #2

19.8

35

0

0

Inoculum Blank Replicate #1

19.8

0

0

0

Inoculum Blank Replicate #2

19.8

0

0

0

Reference Substance

19.8

0

60

0

Abiotic Control

0

35

0

50

Toxicity Control

19.8

35

60

0

Validity criteria fulfilled:
yes
Conclusions:
The NOEC for STP microorganisms is 35 mg/L.
Executive summary:

The toxicity to STP microorgansisms is assessed based on the substance being readily biodegradable in a biodegradation screening study according to OECD TG 301F (Manometric Respirometry) and in compliance with GLP criteria. In this study 35 mg/L test substance was inoculated with activated sludge from a domestic wastewater for 28 days under aerobic conditions. During the incubation period the biological oxygen demand (BOD) was measured and biodegradation expressed as percentage of the theoretical uptake (ThOD). Results for this experiment showed that there was 66% mean biodegradation in the bottles containing test substance on day 28. For the reference substance and toxicity control groups, biodegradation was higher in the toxicity control (containing test substance and reference substance) than in the reference substance alone. On day 28, there was 124% biodegradation in the toxicity control and 82% in the reference substance alone. The 10-day window criterion was not fulfilled (12 % biodegradation on day 8 and 51 % on day 18). The substance is a mono-constituent according to REACH substance identity criteria but consists of more than one stereoisomers and therefore the 10-day window does not need to be met. Based on these findings the substance is assessed as readily biodegradable. According to REACH guidance R.7b, if a compound degrades well in a ready biodegradability test, the tested concentration can be can be used as a NOEC value. Therefore, the NOEC from this study is determined at 35 mg/L.

Description of key information

The toxicity to STP microorgansisms is assessed based on the substance being readily biodegradable in a biodegradation screening study according to OECD TG 301F (Manometric Respirometry) and in compliance with GLP criteria. In this study 35 mg/L test substance was inoculated with activated sludge from a domestic wastewater for 28 days under aerobic conditions. During the incubation period the biological oxygen demand (BOD) was measured and biodegradation expressed as percentage of the theoretical uptake (ThOD). Results for this experiment showed that there was 66% mean biodegradation in the bottles containing test substance on day 28. For the reference substance and toxicity control groups, biodegradation was higher in the toxicity control (containing test substance and reference substance) than in the reference substance alone. On day 28, there was 124% biodegradation in the toxicity control and 82% in the reference substance alone. The 10-day window criterion was not fulfilled (12 % biodegradation on day 8 and 51 % on day 18). The substance is a mono-constituent according to REACH substance identity criteria but consists of more than one stereoisomers and therefore the 10-day window does not need to be met. Based on these findings the substance is assessed as readily biodegradable. According to REACH guidance R.7b, if a compound degrades well in a ready biodegradability test, the tested concentration can be can be used as a NOEC value. Therefore, the NOEC from this study is determined at 35 mg/L.

The NOEC value is determined at 35 mg/L based on the substance being readily biodegradable at this concentration in a biodegradation screening study.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
35 mg/L

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