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Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-04-02 to 2013-07-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guidline Study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD 302 B (Zahn-Wellens Test)
Principles of method if other than guideline:
Toxicity control of the inherent biodegradability test is used to characterize the toxicity of the compound towards aquatic micro-organisms.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material: Not applicable
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
Stock solutions
I. Solution a:
Potassium dihydrogenephosphate (KH2PO4): 8.5 g,
Di-potassium hydrogenephosphate (K2HPO4): 21.75 g,
Sodium di-hydrogenephosphate hydrate (NaH2P04*H20): 33.4 g,
Ammonia chloride (NH4CI): 0.5 g,
H2O demin. ad.: 1000 mL. The pH was adjusted to 7,4 ±0,1.

II. Solution b:
Calcium chloride dihydrate (CaCI2*2H20): 36.4 g,
H2O demin. ad.: 1000 mL.

III. Solution с:
Magnesium sulfate heptahydrate (MgS04*7H20): 22.5 g,
H2O demin. ad.: 1000 mL.

IV. Solution d:
Iron(lll) chloride hexahydrate (FeCI3*6H20): 0.25 g,
Di-sodium-ethylendiamintetraacetate dihydrate (Na2EDTA*2H20): 0.4 g,
H2O demin.: ad 1000 mL.

Test medium
The medium was freshly prepared. Composition:
Solution a: 1 mL,
Solution b: 1 mL,
Solution с: 1 mL,
Solution d: 1 mL,
H2O demin. ad.: 1000 mL

Test Vessels and Apparatus:
Erlenmeyer flasks (nominal volume 2000 mL) were used. For aeration, glass tubes which reach to 2 cm above the bottom of the respective flask, were used. The air was purified by activated charcoal and moistened. An orbital shaker was used for shaking.

Instruments and Devices:
The following instruments and devices were used in the performance of the study:
- Data logger for temperature
- Analytical scales Mettler Toledo XS 205 DU LAUS No. 2
- Precision scales Mettler Toledo XS 6001S
- Adjustable pipettes with one-way tips Rainin®; LAUS-Nos. 29; 40
- Carbon analyser TOC multi N/C 2100S, Analytik Jena
- pH-meter3310 wtw
- Heating chamber Memmert LAUS No. 4
- Orbital shaker GFL3019 LAUS No. 11
- Fridge LAUS No. 9 and 11
- Standard laboratory glassware
- Syringe filters nylon 0.45 pm
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Effluent from a biologic sewage treatment plant was used. The chosen plant is treating mostly household sewage. The effluent was taken from the effluent channel of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf, Germany.
- Storage conditions: The effluent was kept aerobic during transport and storage.
- Storage length: Date of collection: 03. April 2013, batch no: 20121010.
- Pretreatment: The sludge was washed with tap water twice, then filtrated through a cloth and washed and resuspended in test medium. It was then aerated. The dry matter was determined as 4100 mg suspended solids/L.
- Concentration of sludge: concentration 601 mg dry matter/L
- Initial cell/biomass concentration: Inoculum was added in order to reach a content of 600 mg dry matter/L in the final volume
- Water filtered: Yes
- Type and size of filter used, if any: Not reported
Test type:
other: Biodegradation study (OECD Guideline 302B)
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
112 d
Remarks on exposure duration:
Standard test duration (28 d) was prolonged to 112 days to observe ultimate biodegradability of the compound
Post exposure observation period:
None
Test temperature:
The test was performed at room temperature (18.7 - 26.0 °С) without direct lighting.
pH:
With anorganic acid or base, the pH was adjusted to 7.0 ± 0.5.
Details on test conditions:
No further details available
Reference substance (positive control):
yes
Remarks:
A stock solution containing 2.0005 g/L was prepared and TOC was measured in filtrated and unfiltrated solution. The TOC of the unfiltrated solution was 1483.5 mg/L, corresponding to 74.2 % organic carbon.
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
306.2 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
other: As degradation in the toxicity flask was 50 % on day 14, the test item N-Butylpyrrolidone can be stated as "not toxic towards the inoculum in a concentration of 306.2 mg/L".
Details on results:
No further details available
Results with reference substance (positive control):
A stock solution containing 2.0005 g/L was prepared and TOC was measured in filtrated and unfiltrated solution. The TOC of the unfiltrated solution was 1483.5 mg/L, corresponding to 74.2 % organic carbon. Degradation of the positive control was 98% after 8 days.

A Zahn-Wellens Test was performed with n-Butylpyrrolidone to characterise inherent biodegradation potential of the compound. The test item was tested using a concentration of 200 mg organic carbon/L (nominal) in test medium following OECD 302B/EU-Guideline C.9-C. Aniline was chosen as positive control. Activated sludge from a sewage treating plant was used as inoculum (concentration 601 mg dry matter/L). The test was left running for 112 days (sponsor's intent). To measure the degradation of test item and positive control, the content of dissolved organic carbon (DOC) in the test vessels was measured twenty times during the test. All validity criteria were met. The following data could be determined for the test item n-Butylpyrrolidone: The degree of biodegradation reached 7 % after 28 days. Mean degradation at the end of the test (112 days) was 81 % (lag-phase day 0 - 35, degradation phase day 36 -112).

Validity criteria fulfilled:
yes
Conclusions:
As degradation in the toxicity flask was 50 % on day 14, the test item n-Butylpyrrolidone can be stated as "not toxic towards the inoculum in a concentration of 306.2 mg/L".
Executive summary:

Toxicity control of the inherent biodegradability test is used to characterize the toxicity of the compound towards aquatic micro-organisms.

As degradation in the toxicity flask was 50 % on day 14, the test item n-Butylpyrrolidone can be stated as "not toxic towards the inoculum in a concentration of 306.2 mg/L". Degradation of the positive control (aniline) was 98% after 8 days.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-04-12 - 2017-10-03
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 (Carbon and Ammonium Oxidation))
Version / remarks:
Guidelines for Testing of Chemicals, No. 209. Adopted 22 July 2010.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Test Monitoring
The pH of the sludge mixture was measured before sampling for the respiration measurement. Following three hours of aeration, a 23-mL aliquot of each sludge mixture was transferred to a 50-mL cylinder which was placed inside a 20 ± 2 °C water bath placed over a stir plate. The cylinders received a stir bar and were fitted with a dissolved oxygen probe after receiving the sample. The probes were attached to a Strathkelvin Instruments Model 928 oxygen system and a computer that ran the Strathkelvin software (Strathkelvin, 2005). The Strathkelvin system was used to monitor the depletion of dissolved oxygen of six samples over time. Immediately after the dissolved oxygen probes were attached to the Strathkelvin system, the computer program was initiated so that oxygen depletion rates were measured starting at time zero. The oxygen depletion rate was terminated after a 10-minute period. The sludge mixtures were stirred continuously with zero headspace while the oxygen measurements were taken.
The temperature of the environmental chamber in which the test was conducted was monitored using a minimum/maximum thermometer. Solution pH was measured with a Yellow Springs Instrument (YSI) Model pH100 pH meter. The SI Multisample Activated Sludge Respirometer was used for determination of dissolved oxygen consumption. The oxygen depletion rate was measured in every sample from each test system.
Vehicle:
yes
Remarks:
purified water
Details on test solutions:
Standard Reagents
All solutions were prepared using purified reagent water meeting ASTM Type II requirements purified with a Millipore Milli-Q® Direct 8 water purification system. The purified reagent water typically shows greater than 16.7 Mohm-cm resistivity and less than 1 mg/L total organic carbon, which is the established detectable limit at this laboratory. All chemicals used were at least reagent grade and obtained from commercial sources.

Test Substance Stock Solution Preparation
A 4.0 mg/mL primary stock solution was prepared by bringing 4.0108 g (3.999 g as active ingredient) of the test substance to a final volume of 1000 mL with purified reagent water. The stock solution was stored in a refrigerator in a glass volumetric flask fitted with a glass stopper until used on that day. This primary stock solution was used to dose each of the five test solution concentrations.

3,5-Dichlorophenol Stock Solution Preparation
A 500 mg/L 3,5-dichlorophenol stock solution was prepared by first placing 0.2561 g of the reference substance (0.2500 g as active ingredient) in 5.0 mL of 1 N sodium hydroxide (NaOH). Then 10 mL of purified reagent water was added to the solution. While on a stir plate, stirring with a Teflon stir bar, approximately 3.5 mL of 1.0 N sulfuric acid (H2SO4) was slowly added to the solution until a precipitate was observed. The solution was then transferred to a 500-mL volumetric flask and brought to volume with purified reagent water. Finally, the solution was transferred to an amber glass bottle and the pH of the resultant 3,5-dichlorophenol stock solution was 11.28, which required further adjustment with 1 N sulfuric acid (H2SO4) to a final pH of 7.49.

Dilution Water
The dilution water, also called laboratory well water, was a mixture of unadulterated water from a 100-meter bedrock well and dechlorinated Town of Wareham well water. Total hardness, total alkalinity, pH and conductivity were measured weekly from a central location within the laboratory. Hardness and alkalinity were determined according to Standard Methods for the Examination of Water and Wastewater (APHA et al., 2005).
Representative samples of the town water were analyzed biannually for the presence of pesticides, PCBs, and toxic metals by GeoLabs, Inc., Braintree, Massachusetts, in agreement with ASTM standard practice (2007). None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed.
Test organisms (species):
activated sludge
Details on inoculum:
Inoculum Preparation
Prior to test initiation, approximately 16 L of activated sludge was obtained from the New Bedford Waste Water Treatment Plant (WWTP), New Bedford, Massachusetts, which receives primarily domestic sewage. Before use, the sludge solution was passed through a 2-mm stainless steel sieve, concentrated by centrifuging for 10 minutes at 1000 rpm, and the resulting supernatant was poured off. The sludge was then washed with approximately 250 mL of laboratory well water and centrifuged at 1000 rpm for 10 minutes, for a total of four times, with the supernatant being discarded each time. The percent moisture content of the concentrated sludge was obtained with a Sartorius MA-150 moisture balance. The percent moisture was used to calculate the quantity of sludge required for suspension in water to obtain an activated sludge inoculum containing a solids level of 3 grams per liter (± 10%). Based on the percent moisture content of 95.08%, 335.37 g of the sludge was added to 5161.88 mL of laboratory well water to create the first batch of inoculum. The inoculum received 2.75 mL of an undiluted synthetic sewage feed (Table 1) and was aerated overnight until use. A second batch of inoculum was prepared by adding 213.41 g of the same sludge to 3284.84 mL of laboratory well water. The inoculum received 1.75 mL of an undiluted synthetic sewage feed (Table 1) and was aerated overnight until use. Both the first and second batches inoculum were prepared one day before test initiation and had a pH value of 7.21. The solids content of the first batch was determined to be 3.22 g/L and the solids content of the second batch of inoculum was 3.18 g/L.
Test type:
not specified
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
not specified
Test temperature:
20 ± 2 °C
pH:
Both the first and second batches inoculum were prepared one day before test initiation and had a pH value of 7.21.
Dissolved oxygen:
not specified
Salinity:
not specified
Conductivity:
not specified
Nominal and measured concentrations:
Test Concentration Selection
Based on preliminary testing and after consultation with the Study Sponsor, nominal concentrations of 3.0, 9.6, 30.7, 98.3, and 315 mg/L were selected for definitive testing.
Details on test conditions:
Test System
The test was conducted in 1-L glass beakers, which were cleaned prior to use by washing with detergent and water followed, by a 50% nitric acid rinse and finally purified reagent water. The test beakers were labeled with the study number, date, initials, “T.S.” for test substance or “Ref” for reference substance, followed by the concentration value and replicate designation as the identification number. During the contact time (the time the microorganisms were in contact with the substance or 3,5-dichlorophenol prior to the measurement of oxygen consumption; three hours), the solutions were aerated using a Welch air pump delivering room air at a rate of 0.5 to 1.0 L/min. A Hewlett Packard flow meter was used to measure flow rates. The test was conducted at 20 ± 2 °C.

Preparation of Test Solutions
At time 0, 16 mL of synthetic sewage feed was added to a 500-mL graduated cylinder and then brought to a volume of 250 mL with laboratory well water. The microbial inoculum was added until the solution reached a final volume of 500 mL. This mixture was added to a 1-L test beaker and aeration was immediately initiated. This test beaker was identified as Control 1 (C1). An abiotic control was prepared with the substance, synthetic sewage feed, and laboratory well water with no microbial inoculum. The reference substance (3,5-dichlorophenol) test systems were prepared by adding appropriate volumes of the 3,5-dichlorophenol stock solution to the sludge mixture. Control 2 (C2) was prepared in the same manner as Control 1, fifteen minutes after the last reference substance test system was prepared. Lastly, test substance systems were prepared at each substance-concentration by adding appropriate amount of the of the test substance to the sludge mixture. Five replicates were prepared at each test substance concentration. One additional control was initiated with each series of test substance concentrations following the same procedure as Control 1 (C1). All test and reference substance concentrations were based on nominal levels.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
315 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 315 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Throughout the test, the temperature in the chamber where aeration was performed ranged from 19.7 to 20.3 ºC. The temperature of the respirator water bath ranged from 20.9 to 21.9 °C. The air flow measurements across all vessels ranged from 0.5 to 1.0 L/minute.
The results (pH, respiration rate, and percent inhibition) for all test vessels are presented in Table 2. The measured pH value in the abiotic control was 7.35 and the remaining pH values ranged from 7.58 to 7.85. At test termination, the minimum respiration rate and the maximum respiration rate for the six control vessels was determined to be 26.2 and 27.8 mg O2/L/hr, respectively, which met the acceptability criterion (i.e., within 30% of each other during the test).
The mean respiration rate in the test substance test vessels (3.0, 9.6, 30.7, 98.3, and 315 mg/L) was 23.0, 22.1, 28.5, 22.9, and 27.0 mg O2/L/hr, respectively. Compared to the mean of the six controls, the respiratory inhibition for the test substance vessels was 14.9, 18.2, -5.6, 15.3, and 0.1% respectively.
Based on the results, the No-Observed-Effect Concentration (NOEC) was determined to be 315 mg/L by statistical analysis. The EC10 value was empirically estimated to be > 315 mg/L based on these results.
The respiration rate in the 3,5-dichlorophenol test vessels (1.0, 3.0, 10, and 30 mg/L) was 25.8, 25.8, 16.2, and 5.9 mg O2/L/hr, respectively. Compared to the mean of the six controls, the respiratory inhibition for the 3,5-dichlorophenol test vessels was 4.6, 4.6, 40.1, and 78.2%, respectively. Based on the results, the EC50 value for 3,5-dichlorophenol was calculated by linear regression using an Excel computer program and was determined to be 12.3 mg/L, which was inside the acceptable limits as specified in OECD 209 Guideline (i.e., 2 to 25 mg/L).
Results with reference substance (positive control):
The mean respiration rate in the 3,5-dichlorophenol test vessels (1.0, 3.0, 10, and 30 mg/L) was 25.8, 25.8, 16.2, and 5.9 mg O2/L/hr, respectively. Compared to the average of all six controls, respiratory inhibition for the 3,5-dichlorophenol test vessels was 4.6, 4.6, 40.1, and 78.2%, respectively. Based on these results, the EC50 value for 3,5-dichlorophenol was calculated to be 12.3 mg/L, which was within the acceptable limits as specified in the OECD 209 Guideline (i.e., 2 to 25 mg/L).

PROTOCOL DEVIATION

The protocol and the 2010 OECD 209 Guideline states that the oxygen uptake rate for the blank controls should be at least 20 mg O2/g of activated sludge (dry weight of suspended solids). In this study, the oxygen uptake rate for the blank controls was 18 mg O2/g of activated sludge.

Based on the experience of our laboratory, a typical oxygen uptake rate is slightly under 20 mg O2/g of activated sludge. Also, based on the question & answer guidance from Germany (January 13, 2012), this acceptability level may be too stringent for many sources of activated sludge. It is also stated, that as long as the other portions of the test are valid such as the reference compound meeting its acceptability criteria, the study is valid. In the case of this study, all other acceptability criteria were met and the oxygen uptake value was only 10% lower than the acceptable level.

Therefore, this deviation will not negatively impact this study.

Table 2: The pH, respiration rate, percent inhibition, and EC50 value determined in all test vessels during the activated sludge respiration inhibition test

Sample Type pH Respiration Rate (mg O2/L/Hr) % Inhibition
Abiotic 7,35 -0,80 NA = not applicable
Control 1 7,72 27,10 NA
Control 2 7,75 27,80 NA
Control 3 7,78 27,70 NA
Control 4 7,65 26,20 NA
Control 5 7,74 26,60 NA
Control 6 7,74 26,80 NA
3,5 dichlorophenol (mg/L)
1,00 7,76 25,80 4,60
3,00 7,85 25,80 4,60
10,00 7,84 16,20 40,10
30,00 7,78 5,90 78,20
EC50: 12.3 mg 3,5-dichlorophenol/L
Test-substance (mg/L)
3,00 7,73 27,30 -1,00
3,00 7,72 15,40 43,00
3,00 7,74 29,40 -8,80
3,00 7,76 30,30 -12,10
3,00 7,75 12,60 53,40
Mean (SD = Standart deviation) 14,9 (30,9)
9,60 7,67 26,50 2,00
9,60 7,68 11,40 57,80
9,60 7,68 28,90 -6,90
9,60 7,67 27,30 -1,00
9,60 7,71 16,50 39,00
Mean (SD) 18,2 (28,6)
30,70 7,68 27,60 -2,10
30,70 7,70 30,80 -13,90
30,70 7,71 27,70 -2,50
30,70 7,69 27,40 -1,40
30,70 7,69 29,20 -8,00
Mean (SD) -5,6 (5,4)
98,30 7,69 25,60 5,30
98,30 7,69 29,10 -7,60
98,30 7,61 25,60 5,30
98,30 7,61 25,10 7,20
98,30 7,62 9,10 66,30
Mean (SD) 15,3 (29,1)
315,00 7,63 22,90 15,30
315,00 7,59 29,50 -9,10
315,00 7,59 26,50 2,00
315,00 7,58 28,30 -4,70
315,00 7,66 27,80 -2,80
Mean (SD) 0,1 (9,4)
Validity criteria fulfilled:
yes
Conclusions:
At the termination of the test, the respiration rate for all of the control vessels was within 30% of each other during the test. Mean respiration rate in the test substance test vessels (3.0, 9.6, 30.7, 98.3, and 315 mg/L) was 23.0, 22.1, 28.5, 22.9, and 27.0 mg O2/L/hr, respectively. Compared to controls (mean of six values), the respiratory inhibition for the test substance test vessels was 14.9, 18.2, -5.57, 15.3, and 0.1% respectively. Based on these results, the No-Observed-Effect Concentration (NOEC) was determined by statistical means to be 315 mg/L. The EC10 was determined by statistical means to be > 315 mg/L.
Executive summary:

The objective of this study was to determine the effect of test-sample on sludge microorganisms by measuring the respiration rate under defined conditions at different concentrations of the test substance. The use of a reference substance, 3,5-dichlorophenol, in a parallel test allowed for an evaluation of the sensitivity of the microbial population and the adequacy of test procedures. The study was conducted according to GLP and OECD 209.

At the termination of the test, the respiration rate for all of the control vessels was within 30% of each other during the test. Mean respiration rate in the test substance test vessels (3.0, 9.6, 30.7, 98.3, and 315 mg/L) was 23.0, 22.1, 28.5, 22.9, and 27.0 mg O2/L/hr, respectively. Compared to controls (mean of six values), the respiratory inhibition for the test substance test vessels was 14.9, 18.2, -5.57, 15.3, and 0.1% respectively. Based on these results, the No-Observed-Effect Concentration (NOEC) was determined by statistical means to be 315 mg/L. The EC10 was determined by statistical means to be > 315 mg/L.

Description of key information

Toxicity control of the inherent biodegradability test is used to characterize the toxicity of the compound towards aquatic micro-organisms.

As degradation in the toxicity flask was 50 % on day 14, the test item N-butylpyrrolidone can be stated as "not toxic towards the inoculum in a concentration of 306.2 mg/L"

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
306.2 mg/L

Additional information

key, Muckle-2013b

Toxicity control of the inherent biodegradability test is used to characterize the toxicity of the compound towards aquatic micro-organisms.

As degradation in the toxicity flask was 50 % on day 14, the test item N-butylpyrrolidone can be stated as "not toxic towards the inoculum in a concentration of 306.2 mg/L"

key, Riggs-2017

The objective of this study was to determine the effect of test-sample on sludge microorganisms by measuring the respiration rate under defined conditions at different concentrations of the test substance. The use of a reference substance, 3,5-dichlorophenol, in a parallel test allowed for an evaluation of the sensitivity of the microbial population and the adequacy of test procedures. The study was conducted according to GLP and OECD 209.

At the termination of the test, the respiration rate for all of the control vessels was within 30% of each other during the test. Mean respiration rate in the test substance test vessels (3.0, 9.6, 30.7, 98.3, and 315 mg/L) was 23.0, 22.1, 28.5, 22.9, and 27.0 mg O2/L/hr, respectively. Compared to controls (mean of six values), the respiratory inhibition for the test substance test vessels was 14.9, 18.2, -5.57, 15.3, and 0.1% respectively. Based on these results, the No-Observed-Effect Concentration (NOEC) was determined by statistical means to be 315 mg/L. The EC10 was determined by statistical means to be > 315 mg/L.

Value from key, Muckle-2013b was chosen for CSA because this value is a little smaller than that derived from key, Riggs-2017. So a worst-case-approach was chosen, because the smaller value induces the higher environmental hazard.