Niobium

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Jun - 25 Jun 2021

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:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: direct addition on a weight basis: The test item was directly weighed to Teflon pieces, which were subsequently added to the dilution water. To achieve a maximum dilution of the test item, the assay was stirred overnight but for a maximum of 24 h (following the indications for poor water-soluble substances in the "Leitfaden fur Meldungen neuer Stoffe nach dem Chemikaliengesetz"). To ensure the highest possible concentration in the required pH range according to the OECD guideline 209 (7.5 ± 0.5), before use the pH of the mixture including all ingredients except of the inoculum was checked and adjusted as close to 8.0 (actually 7.8 - 7.9) as possible using NaOH (1N) solution.
- Differential loading: 10 (n=1), 100 (n=1), 1000 (n=3) mg/L test item (nominal), with inoculum
- Test concentration separation factor: 10
- Controls: Negative control: 0 mg/L test item ( n=6), with inoculum; Abiotic control: 1000 mg/L test item, without inoculum (n=1); Positive control: 1, 6, 36 mg/L (n=1) 3,5-dichlorophenol, with inoculum

Reference:
Leitfaden fuer Meldungen neuer Stoffe nach dem Chemikaliengesetz. Sechste Oberarbeitete Auflage (2005). Deutsche Bundesanstalt fuer Arbeitsschutz und Arbeitsmedizin, Fachbereich 5, Chemikalien, Anmeldung und Zulassung.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany
- Sampling date: 24th June 2021
- Method of cultivation: On return to the laboratory, the sludge was sieved (2 mm), left alone to settle and the supernatant was decanted. The sludge was washed two times with isotonic solution. A small amount of the sludge was weighed and dried. From this result a stock solution of activated sludge in tap water was prepared with a mixed liquor suspended solids level of about 2 - 4 g dry mass sludge/liter (3.3 g/L). The sludge stock solution was fed with synthetic sewage feed (25 mL/L, according to OECD 209, pH measured: 7.5 +/- 0.5) and was incubated and aerated overnight at 20 ± 2 °C in the dark.
- Preparation of inoculum for exposure: Inoculum was diluted to 1.5 g dry mass sludge/L
- Initial biomass concentration: 1.5 g dry mass sludge/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

19.5 - 20.5 (mean 20.1) °C

pH:

Test start: 7.8 - 7.9
Test end: 8.6 - 8.7 (except for abiotic control: 7.7)

Nominal and measured concentrations:

Nominal: 0, 10, 100, 1000 mg/L test item

Details on test conditions:

TEST SYSTEM
- Test vessel: 1 L Erlenmeyer flasks
- Fill volume: 500 ml
- Aeration: yes, 0.5 - 1 L/min
- Stirring: yes
- No. of vessels per concentration (replicates): n=1 except for 1000 mg/L test item concentration (n=3)
- No. of vessels per control (replicates): n=6, 3 replicates were measured at test start and three at the end of the test
- No. of vessels per positive control (replicates): n=1 per concentration
- No. of vessels per abiotic control (replicates): n=1
- Sludge concentration (weight of dry solids per volume): 1.5 g dry mass sludge/L
- Nitrification inhibitor used (delete if not applicable): none

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, prior to use the pH of the mixture (without inoculum) was adjusted as close to 8.0 as possible using NaOH solution (1 N)
- Photoperiod: dark

EFFECT PARAMETERS MEASURED:
- Respiration rates were measured and recorded after 3 h of incubation over a period of 5-10 minutes or until an O2-content of 2.5 mg O2/L was reached
- Measurements were carried out in glass beakers
- Sensor: O2-sensor (FDO® 925 combined with an inoLab Multi 9630 measuring device by WTW Weilheim)
- pH: at start and end of the test in each test vessel

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

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:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Blank controls oxygen uptake rate: 20.47 mg O2/(L*h) after three hours (mean, n=6)
- Coefficient of variation of oxygen uptake rate in control replicates: 5.9%

Results with reference substance (positive control):

EC50 (3 hours): 16.8 mg/L, this is within the accepted range of an EC50 (3 hours) of 2 to 25 mg/L

The total microbial respiration rate of the activated sludge incubated with concentrations/loadings of 1 to 36 mg 3,5-DCP per liter was found to be at mean within 4.09 and 21.88 mg O2/(L*h) after three hours.

Mean inhibition (%) of the respiration rate by the reference item:
1 mg/L: -6.9%
6 mg/L: 12.9%
36 mg/L: 80%

Reported statistics and error estimates:

A LOEC/NOEL was calculated by using ANOVA followed by Dunnett’s or Williams' test or an appropriate non-parametric test. For trend analysis, a Contrasts test (Monotonicity of Concentration/Response) for metric data was applied. Since the test results of the reference substance show a concentration-response relationship, they were statistically analysed to determine EC values together with 95% confidence intervals using Probit-analysis assuming log-normal distribution of the values. For statistical calculations, the computer program ToxRat Professional version 3.3.0 by ToxRat® Solutions GmbH was applied.

The total microbial respiration rate of the activated sludge incubated with concentrations/loadings of 10 to 1000 mg Niobium Metal Powder per liter and in the controls without test item was found to be within 20.5 and 23.7 mg O2/(L*h) after three hours. The respiration rate was not inhibited by the test item.

Table 1: Validity criteria OECD 209 (2010)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.	20.47 mg O2/g activated sludge (dry weight of suspended solids) in an hour	yes
The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.	5.9%	yes

Validity criteria fulfilled:

yes

Remarks:

For details please refer to field "any other information on results incl. tables"

Description of key information

Toxicity to microorganisms:

EC50 (3 h) > 1000 mg/L (nominal) for microorganisms of activated sludge (OECD 209, limit test)

NOEC (3 h) ≥ 1000 mg/L (nominal) for microorganisms of activated sludge (OECD 209, limit test)

Additional information

The available key study on toxicity to activated sludge was performed according to OECD guideline 209 (GLP) by exposing microorganisms of freshly collected activated sludge, from a mainly domestic German sewage treatment plant, to the test substance for 3 h. In an combined range finding/limit test, exposure occurred to 0, 10, 100 and 1000 mg/L of the test substance. The highest water solubility of the test substance was expected at pH 8.5. Therefore, the test item mixture including all ingredients except the inoculum was adjusted as close as possible to the maximum acceptable pH value of 8.0 according to OECD guideline 209. No concentration dependent effects on the total respiration rate of the microorganisms of the activated sludge were observed after 3 h up to the highest concentration tested, resulting in an EC50 of > 1000 mg/L (nominal) and a NOEC value of ≥ 1000 mg/L (nominal).