Niobium hydride

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:

11 July 2018 to 13 July 2018

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

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- The test material was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore weighed amounts were added to the amber test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). The test item – Milli-RO water mixtures were magnetically stirred for a short period. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required concentrations. Optimal contact between the test material and test organisms was ensured by applying continuous aeration and stirring.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

-Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of the sludge: 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 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 initial pH of the sludge was 5.6 on the day of testing. The sludge was buffered to a pH of 6.4 using sodium bicarbonate solution (70 g/L). Before the sludge was used, the respiration rate of the sludge was determined to verify the quality of the sludge. The mean respiration rate was 26 mg O2/g.h and within the validity criterion.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 - 21 °C

pH:

6.8 - 7.5

Nominal and measured concentrations:

Nominal: 10, 100 and 1 000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass open bottles/vessels
- Material, size, headspace, fill volume: 500 mL
- Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70 % saturation (60 % of air saturation is > 5 mg/L at 20 °C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): The highest concentration was tested in triplicate, lower concentrations consisted of one replicate.
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 3.0 g/L
- Nutrients provided for bacteria: Synthetic medium (=sewage feed): 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O and 2.8 g K2HPO4. Dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.
- The synthetic medium (= sewage feed, 16 mL) and an a appropriate amount of the test material stock were mixed and made up to 250 mL with Milli-RO water. The pH was determined. Thereafter, 250 mL activated sludge was added. This was the start of the test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO-medium, formulated using RO-water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition: CaCl2.2H2O 211.5 mg/L, MgSO4.7H2O 88.8 mg/L, NaHCO3 46.7 mg/L and KCl 4.2 mg/L.

EFFECT PARAMETERS MEASURED
- 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.
- The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference material concentrations and controls. The medium temperature was recorded continuously in a temperature control vessel. The temperature control vessel was identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
- Oxygen recording: Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

TEST CONCENTRATIONS
- Combined Limit/Range-Finding Test: In a combined limit/range-finding test concentrations of 10, 100 and 1 000 mg/L were tested. The highest concentration was tested in triplicate, lower concentrations consisted of one replicate. In addition, a control (6 replicates) and an abiotic control (1 replicate) were tested.

ANALYSIS
Calculations
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

- Calculation of oxygen uptakes: The respiration rate (R) 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.

R was calculated by the BlueBox software as (V1 – V2)/Δt * 60

Where:
V1 = Value 1: the oxygen concentration at the start of the selected section of the linear phase (mg O2/L),
V2 = Value 2: the oxygen concentration at the end of the selected section of the linear phase (mg O2/L),
Δt is the time interval between these two measurements.
Negative R values were expressed as 0 mg O2/L.h (V1
Furthermore the respiration rate was expressed as the amount of oxygen consumed per g dry weight of sludge per hour (Rs in mg O2/g.h).

Rs = R / SS

Where:
SS is the concentration of suspended solids in the test mixture (g/L).

- Calculation of the inhibition of the respiration rate: The percentage inhibition, IT, of total oxygen consumption is given below:

IT = [1- (RT/RC)] x 100 %

Where:
RT is the respiration rate of the test material (T) (mg O2/L.h or mg O2/g.h).
RC is the respiration rate of the control (C) (mg O2/L.h or mg O2/g.h).
In case abiotic uptake was observed (>5 % of control respiration) a correction for this oxygen uptake will be made.

- Interpretation of results:
ECx:
- For the reference material, calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference material.
- For the test material, no EC50 value could be calculated because effects were below 50 % (EC50 > 1 000 mg/L).

NOEC determination:
- An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Two-sample t-test Procedure, α=0.05, one-sided, smaller).
- Calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

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

Remarks:

respiration rate

Details on results:

INHIBITION OF RESPIRATION RATE
- The combined limit/range-finding test showed 20 %, 19 % and an average of 16 % inhibition of the respiration rate at 10, 100 and 1 000 mg/L, respectively. Therefore, the EC50 was above the highest concentration tested (1 000 mg/L).
- In the present test the test material was slightly toxic to waste water (activated sludge) bacteria at 1 000 mg/L). Therefore, a NOEC could not be determined. As discussed with the sponsor no further testing was performed to determine the NOEC.
- There was no oxygen uptake from abiotic processes.

EXPERIMENTAL CONDITIONS
- The pH in all test vessels, before addition of sludge was 7.5. After the 3-hour exposure period the pH was between 6.8 and 7.5.
- The temperature continuously measured in the temperature control vessel ranged between 20 and 21 °C during the test, and complied with the requirements as laid down in the study plan (20 ± 2 °C).

ACCEPTABILITY OF THE TEST
1. The mean control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (22 mg oxygen per one gram of activated sludge). The coefficient of variation of oxygen uptake in control replicates did not exceed 30 % at the end of the definitive test (11 %).
2. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (2.4 mg/L).
Since all criteria for acceptability of the test were met, this study was considered to be valid.

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (2.4 mg/L).

Table 1: Results - Respiration Rate/Inhibition, pH Values

Replicate	Concentration (mg/L)	pH		Respiration Rate		% Inhibition Respiration Rate (Mean Value)
		Start	End	(mg O2/L.h)	(mg O2/g.h)¹
C1	0	7.5	6.9	32.74	21.83
C2	0	7.5	6.9	29.99	19.99
C3	0	7.5	6.8	31.80	21.20
C4	0	7.5	6.8	37.82	25.21
C5	0	7.5	6.8	31.34	20.89
C6	0	7.5	6.8	38.61	25.74
C Mean				33.72	22.48(Rc)
SD				3.60	2.40
CV (%)				11	11
R1	1.0	7.5	7.5	22.12	14.75	34.39
R2	3.2	7.5	7.5	14.91	9.94	55.78
R3	10	7.5	7.3	8.85	5.90	73.75
R4	32	7.5	7.2	0.82	0.55	97.57
T1	10	7.5	6.9	26.95	17.97	20.07
T2	100	7.5	6.9	27.23	18.15	19.24
T3a	1 000	7.5	6.9	28.31	18.87	16.04
T3b	1 000	7.5	6.9	28.21	18.81	16.33
T3c	1 000	7.5	6.8	28.82	19.21	14.52
T3 Mean				28.45	18.96(RT)*	15.63 (IT)
TA	1 000	7.5	7.3	0.00#	0.00	100.0

C: Control

R: Reference item, 3,5-dichlorophenol

T: Test material

TA: Abiotic control of test material

SD: Standard deviation

CV: Coefficient of variation

RT: Total respiration with the test material

RC: Total respiration in the control

IT: % inhibition of total respiration relative to RC

¹ The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control

# No respiration, therefore expressed as 0 mg O2/L.h

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test material was slightly toxic to waste water bacteria (activated sludge) at 1 000 mg/L. The EC50 was above 1 000 mg/L.

Executive summary:

The toxicity of the test material to aquatic microorganisms was investigated in accordance with the standardised guideline OECD 209, under GLP conditions.

The objective of the study was to evaluate the test material for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC50 and/or the no - observed effect concentration (NOEC). The influence of the test material on the respiration rate of activated sludge was investigated after a contact time of 3-hours.

The test material was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test-bottles containing Milli-RO water. The test material–Milli-RO water mixtures were magnetically stirred for a short period. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required concentrations. Optimal contact between the test material and test medium was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

In a combined limit/range-finding test concentrations of 10, 100 and 1 000 mg/L were tested. The highest concentration was tested in triplicate, lower concentrations consisted of one replicate. Furthermore, at 1 000 mg/L an abiotic control (1 replicate) was tested. Responses were compared to the controls.

In the present test the test material was slightly toxic to waste water (activated sludge) bacteria at 1 000 mg/L. Therefore, a NOEC could not be determined. As discussed with the Sponsor no further testing was performed to determine the NOEC. There was no oxygen uptake from abiotic processes.

The batch of activated sludge was tested for sensitivity with the reference material 3,5-dichlorophenol, and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the test material was slightly toxic to waste water bacteria (activated sludge) at 1 000 mg/L. The EC50 was above 1 000 mg/L.

Description of key information

Under the conditions of this study, the test material was slightly toxic to waste water bacteria (activated sludge) at 1 000 mg/L. The EC50 was above 1 000 mg/L.

Additional information

The toxicity of the test material to aquatic microorganisms was investigated in accordance with the standardised guideline OECD 209, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of the study was to evaluate the test material for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC50 and/or the no - observed effect concentration (NOEC). The influence of the test material on the respiration rate of activated sludge was investigated after a contact time of 3-hours.

The test material was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test-bottles containing Milli-RO water. The test material – Milli-RO water mixtures were magnetically stirred for a short period. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required concentrations. Optimal contact between the test material and test medium was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

In a combined limit/range-finding test concentrations of 10, 100 and 1 000 mg/L were tested. The highest concentration was tested in triplicate, lower concentrations consisted of one replicate. Furthermore, at 1 000 mg/L an abiotic control (1 replicate) was tested. Responses were compared to the controls.

In the present test the test material was slightly toxic to waste water (activated sludge) bacteria at 1 000 mg/L. Therefore, a NOEC could not be determined. As discussed with the Sponsor no further testing was performed to determine the NOEC. There was no oxygen uptake from abiotic processes.

The batch of activated sludge was tested for sensitivity with the reference material 3,5-dichlorophenol, and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the test material was slightly toxic to waste water bacteria (activated sludge) at 1 000 mg/L. The EC50 was above 1 000 mg/L.