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Endpoint:
activated sludge nitrification inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Appropriate but old publication.
Principles of method if other than guideline:
Method: Investigation of 52 industrial substances for inhibition of ammonia oxidation by nitrifying bacteria.
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data
Analytical monitoring:
yes
Details on sampling:
See below.
Vehicle:
no
Details on test solutions:
no data
Test organisms (species):
other: nitrifying bacteria, Nitrosomonas sp. and Nitrobacter sp.
Details on inoculum:
Throughout these experiments a stock culture of autotrophic nitrifying bacteria was maintained under continuous culture conditions. Organisms were removed from this culture and used in a standard assay to determine the effects of the individual compounds by comparing the rate of nitrate production in the presence of a test compound to the rate in its absence. All determinations were made in duplicate, and a compound was considered to be inhibitory if it reduced the rate by more than 10 percent.

Stock nitrifying culture. The culture of autotrophic nitrifying bacteria was maintained in a 12 L fiber-wall reactor, consisting of a porous, fuzzy nylon bag, supported on a plastic-coated wire mesh inside a 15 L plastic cylinder. The bag acted as an aeration chamber with internal cell recycle. The bag entrapped the organisms but allowed the effluent to pass, thereby eliminating the need for a settler. Waste sludge was removed from the inside of the bag to maintain a solids retention time of 24 days. Mixing and aeration were accomplished by passing filtered and humidified compressed air at 12.5 L/min through submerged diffusers.

Feed solution was delivered continually to the reactor at a rate of 20 L/day by a peristaltic pump. The feed solution (Table III, see attachment) was prepared according to the medium described in "Methods in Microbiology" for nitrifying bacteria with some minor revisions. Sodium carbonate and bicarbonate were used as a buffer to maintain the pH around 8 throughout the research.
Test type:
static
Limit test:
yes
Total exposure duration:
2 h
Remarks on exposure duration:
no data
Post exposure observation period:
no data
Hardness:
For test medium see the attachment.
Test temperature:
no data
pH:
8.1
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
Test substance concentration: 100 mg/L.
NH3-N concentration: 200 mg/L.
Details on test conditions:
Standard assay procedure.
A standard procedure was developed for determining the rate of nitrate production by organisms taken from the stock culture. The procedure included the following steps:
1. The required volume of mixed liquor was removed from the stock nitrifying culture and centrifuged at 18 400 x g for 15 min.
2. The supernatant was discarded and the cells were dispersed in the feed solution buffered to pH 8.1.
3. A 90-mL aliquot of the resultant cell suspension was added to each reaction flask.
4. 10 ml of de-ionized water was added to each control flask, bringing the total volume to 100 mL, and the NH3-N concentration to 200 mg/I.
5. A 10-mL aliquot of a solution containing the compound to be tested was added to each of duplicate flasks, bringing the total volume to 100 mL and the NH3-N concentration to 200 mg/L.
6. The pH of each flask was measured and adjusted to 8.1 if necessary.
7. At t = 5 min a 25-mL aliquot was removed from each flask and immediately filtered by pressure filtration through a membrane filter to remove the cells. The filtrate was put in cold storage for later determination of the concentrations of nitrite and nitrate nitrogen.
8. The flasks were placed on a shaker at 80 oscillations/min.
9. At t = 1 h and at t = 2 h, Step 7 was repeated.
10. At t = 2 h the pH of each flask was measured and recorded.
11. The filtered samples were analyzed for nitrite (naphthylamine-sulfanilic acid method) and nitrate (cadmium reduction-modified naphthylamine-sulfanilic acid method) nitrogen using Hach Chemical Co. chemicals.
12. A plot was made of N03-N versus time for each flask and the rate of nitrate production was calculated with the linear least squares technique. Inhibition effects were determined by comparing the rates in the test flasks with the rates in control flasks containing the same concentration of microorganisms.

With the exception of the presence of the compound being tested, all conditions for the control were identical to those of the test flask.

The procedure outlined does not distinguish between inhibition of Nitrosomonas sp. and Nitrobacter sp. because the results are based on the rate of nitrate production with ammonia as the substrate. Consequently, any compound found to be inhibitory was retested with nitrite as the substrate at a concentration of 100 mg/L as N. The procedure was the same except that the brucine sulfate method was used to determine the rate of nitrate production. Comparison of the results from the two assays allowed determination of the relative inhibition of the two types of organisms.
Prior to the start of the experiments the coefficient of variation of the nitrate test was found to be 2.4 percent over the entire range of expected concentrations. For the nitrite determination the coefficient of variation was 1.7 percent except for the lowest concentration (approximately 0.04 mg/l), for which it was 9.6 percent. It was established experimentally that the rate of nitrate production was linear over the 2-h assay period. By using a linear least squares line of best fit in the standard assay procedure, it was possible to determine the rate of nitrate production with a coefficient of variation of 8.5 percent. Therefore, a test compound was not considered to be inhibitory unless it reduced the rate of nitrate production by more than 10 percent with respect to the controls.

Screening tests.
All compounds were tested for inhibitory effects at a concentration of 100 mg/I. This concentration was chosen because it represents a likely upper limit on the concentration of any individual compound in an industrial waste stream. If no inhibition is caused at that concentration, then there is little likelihood of inhibition occurring in a treatment plant.

Effects of inhibitor concentration.
Those compounds found to be inhibitory at 100 mg/l were retested at concentrations of 50 and 10 mg/l to determine the effects of concentration on the degree of inhibition. In the case of a few compounds, additional concentrations were tested and these are noted in the results.

Characterization of inhibition.
Additional studies were performed with three inhibitors of ammonia oxidation for further characterization of the nature of the inhibition. For each compound tested the degree of inhibition was determined for each of two inhibitor concentrations at four substrate concentrations. The inhibitor concentrations used depended on the compound being studied, and will be presented in the results. The substrate concentrations used were 50, 100, 150, and 200 mg/l of NH3-N. Controls were run for each substrate concentration. Characterization of the inhibition was accomplished by examination of Hunter-Downs plots.

Reference substance (positive control):
yes
Remarks:
3 substances, i.a. aniline
Duration:
2 h
Dose descriptor:
EC0
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Details on results:
Melamine at 100 mg/L did not inhibit ammonia oxidation by Nitrosomonas sp.
Melamine at 100 mg/L did not inhibit nitrite oxidation by Nitrobacter sp.
Results with reference substance (positive control):
Aniline at 1 mg/L caused an inhibition of ammonia oxidation by 50 %.
Reported statistics and error estimates:
Not relevant.
Validity criteria fulfilled:
yes
Conclusions:
The toxicity of melamine to nitrifying bacteria is low.
Executive summary:

Melamine at 100 mg/L did not inhibit ammonia oxidation by Nitrosomonas sp.

Melamine at 100 mg/L did not inhibit nitrite oxidation by Nitrobacter sp.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1988
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Short report without many details of the method used.
Qualifier:
equivalent or similar to
Guideline:
other: DIN 38412, part 27.
Deviations:
not specified
Principles of method if other than guideline:
Pseudomonas putida respiration inhibition test, DIN 38412 Teil 27.
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
not specified
Test organisms (species):
Pseudomonas putida
Test type:
static
Limit test:
no
Total exposure duration:
30 min
Hardness:
A test medium was used.
Test temperature:
22 +- 2 °C
pH:
7.2 - 7.4
Dissolved oxygen:
Cultures were aerated.
Nominal and measured concentrations:
nominal conc.: 10, 5, 1, 0. 1 g/L.
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
> 10 g/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 10 g/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Validity criteria fulfilled:
not specified
Conclusions:
The toxicity of melamine to Pseudomonas putida, as measured by the respiration inhibition, is low.
Executive summary:

The respiration inhibition of Pseudomonas putida by melamine was investigated. The EC0 >10 g/L.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-07-23 - 2015-10-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was directly added to the test medium and subsequently stirred for 24 hours.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Fresh samples of activated sludge were taken from a sewage treatment plant (Ruhrverband Kläranlage, Schmallenberg, Germany) mainly fed with municipal wastewater.
- Pretreatment: the sludge was washed with isotonic solution. A stock solution of activated sludge in tap water was prepared with a mixed liquor suspended solids level of 4.6 g dry mass sludge/liter. The sludge stock solution was fed with synthetic sewage feed (see chapter 4.3) and was
incubated aerated overnight at 20 ± 2 °C in the dark.
- Initial biomass concentration: 1.5 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C.
pH:
8.1 - 8.2
Nominal and measured concentrations:
Nominal test substance concentrations: 10, 100, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask, 1 L
- Material, fill volume: glass, 500 mL
- Aeration: yes
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 6 + 1 abiotic control


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water, free from inhibitory concentrations of toxic substances (e.g. Cu2+ ions)


OTHER TEST CONDITIONS
- Adjustment of pH: yes, buffered to 7.5 ± 0.5 using H2SO4 (50 g/L) solution.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Microbial respiration rate (total oxygen uptake including heterotrophic and nitrification uptake)

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1000 mg/L
- Results used to determine the conditions for the definitive study: EC50 > 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
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
Remarks:
respiration rate
Details on results:
No inhibition of the respiration rate was observed
Results with reference substance (positive control):
EC50: 2 - 25 mg/L.

Inhibition (%) of the respiration rate by the test item

Test item concentration

10 mg/L

100 mg/L

1000 mg/L

Inhibition

-14.1 %

-4.4 %

0.5 %

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
weight of evidence
Justification for type of information:
Please refer to the analogue justification attached to section 13
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Duration:
2 h
Dose descriptor:
EC0
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Remarks on result:
other: Source, WoE, RA-A, CAS 108-78-1, Hockenbury & Grady 1977.
Duration:
0.5 h
Dose descriptor:
EC10
Effect conc.:
> 10 g/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Source, WoE, RA-A, CAS 108-78-1, Tillmann 1990
Duration:
0.5 h
Dose descriptor:
EC50
Effect conc.:
> 10 g/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Source, WoE, RA-A, CAS 108-78-1, Tillmann 1990
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
Remarks:
respiration rate
Remarks on result:
other: Source, WoE, RA-A, CAS 7758-23-8, Fraunhofer, 2015

Description of key information

 Not toxic to STP microorganisms

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
10 g/L

Additional information

Data on the toxicity of the target substance diphosphoric acid, compound with 1,3,5-triamine (1:2) to aquatic algae are not available. The evaluation of the acute toxicity is based on a weight of evidence approach studies conducted with available data on the source substances melamine (CAS 108-78-7) and calcium bis(dihydrogen orthophosphate) (CAS 7758-23-8). Whereby melamine represents the 1,3,5-triamine moiety of the target substance. The source substance calcium bis(dihydrogenorthophosphate) was selected to assess the toxicity of the diphosphoric acid moiety. The read across approach is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. Further justification is given in the analogue justification in IUCLID section 13.

Two tests investigating the effects of the source substance melamine on microorganisms are available. Hockenbury and Grady (1977) investigated possible effects of 52 industrial substances on the ammonia oxidation by nitrifying bacteria. Nitrifying bacteria were exposed to a substance concentration of 100 mg/L for 2 hours. For melamine an EC0 (2 h) >100 mg/L (nominal) was determined. The second available test determined the inhibition of respiration of Pseudomonas putida exposed to test substance concentrations of 0.1, 1, 5, and 10 g/L (Tillmann, 1990). The determined EC10 (30 min) was > 10 g/L (EC50 (30 min) >10 g/L).

Detrimental effects of phosphate on the respiration rate of microorganism are not expected. The assumption is supported by read across data available for the source substance calcium bis(dihydrogenorthophosphate). The effect of calcium bis(dihydrogenorthophosphate) on the respiration of activated sludge was tested in a study following OECD guideline 209. Activated sludge was exposed to nominal substance concentrations of 10, 100 and 1000 mg/L respectively. The oxygen consumption of the activated sludge was observed for 3 hours. An effect on the respiration rate of the activated sludge was not observed. At test termination an EC10> 1000 mg/L (nominal) was determined.