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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 - 24 Mar 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
yes
Remarks:
Temperature was outside the prescribed range (20 ± 2°C). During exposure, the temperature was 16-23°C. Assessment: Validity criteria for controls and reference item were met and therefore this deviation is considered not to have influenced the conclusion.
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
yes
Remarks:
Temperature was outside the prescribed range (20 ± 2°C). During exposure, the temperature was 16-23°C. Assessment: Validity criteria for controls and reference item were met and therefore this deviation is considered not to have influenced the conclusion.
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot 38022
- Expiration date of the lot/batch: 31 May 2017
- Purity: 95.11%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient room temperature
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water, therefore, test substance stock solutions were prepared at nominal concentrations of 2.5 times the final loading rates in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass.,
USA). Solutions were magnetically stirred for approximately 24 hours in closed, dark brown bottles.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's- Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.8 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
15.8 °C - 22.5 °C
pH:
7.2 - 7.4 before addition of sludge
7.7 - 8.2 after 3-hr exposure period
Nominal and measured concentrations:
100, 180, 320, 560 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass bottles/vessels
- Type: open
- Material, size, headspace, fill volume: All glass bottles/vessels, 500 mL fill volume
- Aeration: Yes, to maintain dissolved oxygen at > 5 mg/L
- No. of vessels per concentration (replicates) 5
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 1.5 g/L
- Nutrients provided for bacteria: 16.0g peptone, 11.0 g meat extract, 3.0 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4 (dissolved in 1 L of water), and filtered
- Nitrification inhibitor used (delete if not applicable): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by reverse osmosis
OTHER TEST CONDITIONS
- Adjustment of pH: No
- Details on termination of incubation: After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.


EFFECT PARAMETERS MEASURED: Dissolved oxygen concentration after 3 hours exposure. The respiration rate from each vessel, in mg O2/L.h, was calculated or interpolated from the linear part of the respiration curve, which was generally between 2 and 7 mg O2/L.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: Yes
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
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
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
301 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% confidence interval 193-385 mg/Ll
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 7.7 mg/L The EC50 was within the 2 to 25 mg/L range considered acceptable for the test.
Reported statistics and error estimates:
ECx
For the reference item calculation of the EC50 value was based on non-linear regression analysis (3-parameter logistic Cumulative Distribution Function) with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item. Calculation of ELx values was based on probit analysis using linear maximum likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding loading rates of the test item.
For MTDID 15078 no EC50-value could be calculated because effects were below 50% (EC50 > 1000 mg/L).
NOEC determination
An effect was considered to be significant if statistical analysis of the data obtained for the test loading rates compared with those obtained in the control revealed significant inhibition of the respiration rate (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
Calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1. Respiration Rates and Percent Inhibitions

Treatment/Nominal Concentration

Respiration Rate (mg O2/L/hour)

Percent Inhibition

Control

34.58

NA

3,5-dichlorophenol 1.0 mg/L

36.14

-4.53

3,5-dichlorophenol 3.2 mg/L

23.65

31.60

3,5-dichlorophenol 10 mg/L

16.46

52.39

3,5-dichlorophenol 32 mg/L

2.78

91.96

MTDID 15078 100 mg/L

38.38

-11.01

MTDID 15078 180 mg/L

34.01

1.65

MTDID 15078 320 mg/L

31.09

10.09

MTDID 15078 560 mg/L

26.22

24.17

MTDID 15078 1000 mg/L

22.36

35.33

Validity criteria fulfilled:
yes
Remarks:
Control oxygen uptake rate > 20 mg O2/gram sludge/hour; coeff. of variation < 30% for control replicates; ref. substance EC50 in range (2-25 mg/L)
Conclusions:
The 3-hour EC50 (respiration) of FBSEE diol to activated sludge is > 1000 mg/L. The corresponding 3-hour NOEC is 320 mg/L. The test was conducted according to OECD 209 and ISO 8192 guidelines.
Executive summary:

The toxicity of FBSEE diol to activated sludge was assessed according to the OECD 209 and ISO 8192 guidelines. The validity criteria were met based on an acceptable level of oxygen uptake in control samples, acceptable variation among control replicates and a typical level of inhibition with the positive control. The 3-hour EC50 (respiration) of FBSEE diol to activated sludge is > 1000 mg/L. The corresponding 3-hour NOEC is 320 mg/L. The study was performed in accordance with internationally-accepted test guidelines under GLP. The study is deemed reliable without restriction and suitable for use in Risk Assessment, Classification & Labeling, and PBT Analysis.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
28-June-2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
Deviations:
no
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Physical state: white powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: store away from heat
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Test solutions, 0 (dilution water control) and 1000 mg/L, were prepared by direct addition of the test substance.
Test organisms (species):
activated sludge
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge from the City of Two Harbors Wastewater Treatment Plant, Two Harbors, MN, USA.
- Preparation of inoculum for exposure: Upon arrival at the laboratory, the sludge was washed with test dilution water and aerated at test temperature. The sludge was determined to have an initial suspended solids concentration of 4.90 g/L. The sludge was then thinned with test dilution water to a calculated suspended solids concentration of 4.0 g/L. This mixture was aerated at test temperature until use. The sludge was used within 24 hours of collection. The sludge was fed synthetic sewage feed during holding.
- Initial biomass concentration: The measured total suspended solids concentration in the 1000 mg/L test solution was 1.59 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
20.2 - 20.8 °C
pH:
7.71 - 7.78 initial
7.65 - 8.57 after 3-hr exposure period
Dissolved oxygen:
6.4 - 8.4 mg/L - initial (0.5 minutes)
Nominal and measured concentrations:
Nominal: 0 mg/L, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers
- Type: open
- Material, size, headspace, fill volume: borosilicate, 1 Liter, 500 mL fill volume
- Aeration: Yes, adequate for particulate suspension while minimizing foaming
- No. of vessels per concentration (replicates) 2
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 1.51 g/L, mean, blank, 1.59 g/L, mean, 1000 mg/L test solutions
- Nutrients provided for bacteria: 16.0g peptone, 11.0 g beef extract, 3.0 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4 (dissolved in 1 L of water), and filtered
- Nitrification inhibitor used (delete if not applicable): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated, deionized tap water
OTHER TEST CONDITIONS
- Adjustment of pH: No
- Details on termination of incubation: After the 30-minute and 3-hour contact times, samples were removed to fill 300 mL BOD bottles. Using a YSI 52 oxygen meter with a self-stirring probe, the dissolved oxygen concentration was determined every 0.5 minutes for a period of 5.5 minutes with the first determination made after 0.5 minutes. After the measurement period the subsample was returned to the test vessel.

EFFECT PARAMETERS MEASURED: Dissolved oxygen concentration was measured after 30 minutes and after 3 hours exposure; measured concentrations ranged from 2.7 to 8.4 mg O2/L. For each exposure, dissolved oxygen depletion and time interval were correlated and the slope of the regression line was calculated as the respiration rate.

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
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
Key result
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 on result:
other: 1000 mg/L was the highest, and only concentration of FBSEE diacid evaluated
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 9.7 mg/L The EC50 was within the 5 to 30 mg/L range considered acceptable for the test.
Reported statistics and error estimates:
EC50 calculation: Trimmed Spearman Karber

Treatment/Nominal Concentration Respiration Rate (mg O2/L/min) Percent Inhibition
Control A (30 min) 0.6091 NA
Control B (30 min) 0.6673 NA
FBSEE diacid 1000 mg/L A (30 min) 0.5964 7
FBSEE diacid 1000 mg/L B (30 min) 0.6000 5
Control A (3 hr) 0.6982 NA
Control B (3 hr) 0.7236 NA
FBSEE diacid 1000 mg/L A (3 hr) 0.6836 2
FBSEE diacid 1000 mg/L B (3 hr) 0.7818 -12
3,5-dichlorophenol 5 mg/L (3 hr) 0.5636 20
3,5-dichlorophenol 15 mg/L (3 hr) 0.2036 71
3,5-dichlorophenol 30 mg/L (3 hr) 0.0836 88
Validity criteria fulfilled:
yes
Remarks:
Control oxygen uptake rate > 20 mg O2/gram sludge/hour; coeff. of variation <15% for control replicates; ref. substance EC50 in range (5-30 mg/L)
Conclusions:
The 3-hour EC50 (respiration) of FBSEE diacid to activated sludge is > 1000 mg/L, and the corresponding 3-hour NOEC is 1000 mg/L. The test was a limit test at 1000 mg/L, conducted according to the OECD 209 guideline.
Executive summary:

FBSEE diacid is a degradation product of FBSEE diol. The toxicity of FBSEE diacid to activated sludge was assessed according to the OECD 209 guideline in a limit test conducted at 1000 mg/L. The validity criteria were met based on an acceptable level of oxygen uptake in control samples, acceptable variation among control replicates and a typical level of inhibition with the positive control. The 3-hour EC50 (respiration) of FBSEE diacid to activated sludge is > 1000 mg/L, and the corresponding 3-hour NOEC is 1000 mg/L as no inhibition was was observed. The study followed an international standard method but was not GLP compliant. The results are for a degradation product rather than the parent material. Therefore the study is deemed reliable with restrictions.

Description of key information

The 3-hour EC50 (respiration) of FBSEE diol to activated sludge is > 1000 mg/L. The corresponding 3-hour NOEC is 320 mg/L. The test was conducted according to OECD 209 and ISO 8192 guidelines.

Key value for chemical safety assessment

Additional information

In the key study, the toxicity of FBSEE diol to activated sludge was assessed according to the OECD 209 and ISO 8192 guidelines. The validity criteria were met based on an acceptable level of oxygen uptake in control samples, acceptable variation among control replicates and a typical level of inhibition with the positive control. The 3-hour EC50 (respiration) of FBSEE diol to activated sludge is > 1000 mg/L. The corresponding 3-hour NOEC is 320 mg/L, and the EC10 is 301 mg/L. The study was performed in accordance with internationally-accepted test guidelines under GLP. The study is deemed reliable without restriction and suitable for use in Risk Assessment, Classification & Labeling, and PBT Analysis.

FBSEE diol will be rapidly converted to FBSEE diacid (see section 5.2.1). In the supporting study, the toxicity of FBSEE diacid to activated sludge was assessed according to the OECD 209 guideline in a limit test conducted at 1000 mg/L. The validity criteria were met based on an acceptable level of oxygen uptake in control samples, acceptable variation among control replicates and a typical level of inhibition with the positive control. The 3-hour EC50 (respiration) of FBSEE diacid to activated sludge is > 1000 mg/L, and the corresponding 3-hour NOEC is 1000 mg/L as no inhibition was observed. The study followed an international standard method but was not GLP compliant. The results are for a degradation product rather than the parent material. Therefore the study is deemed reliable with restrictions.