Molybdenum trioxide, reaction products with bis[O,O-bis(2-ethylhexyl)] hydrogen dithiophosphate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Remarks:

(Includes evaluation of heterotrophic and nitrification respiration inhibition.)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 July 2017 to ****

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))

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

Guideline deviation:
The total and heterotrophic respiration inhibition was evaluated using one set of vessels in which oxygen consumption was measured before and after amendment with a nitrification inhibitor (ATU), rather than with two discrete sets of vessels (i.e. with and without ATU) incubated for 3 hours as described in the OECD Guideline 209. This deviation is not considered to have adversely impacted the outcome of the study because it reduces the inherent variability within the test system and the validity criteria for the controls and reference substance were met for all respiration endpoints.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

-Purity: Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
-Description: Dark Green Liquid

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Each treatment vessel contained a synthetic sewage concentrate, dechlorinated tap water, microbial inoculum, test or reference substance (blank controls had neither) and a nitrification inhibitor (for heterotrophic respiration assessment) to achieve total test volumes of 250 mL.

The test substance was weighed into individual foil boats and added directly to test vessels to achieve nominal concentrations of 1, 10 and 100 mg/L (duplicate) and 1000 mg/L (triplicate). Immediately after preparation, the pH of each vessel was determined and adjusted to within 7.5 ± 0.5 g/L with 40% NaOH, as necessary.

The reference substance, 3,5-dichlorophenol (3,5-DCP), was added the test vessels as a prepared aqueous stock solution (0.50 g/L) to achieve nominal concentrations of 0.1, 2.0 and 40 mg/L (single vessel for each).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: sludge return line at Burley Menston sewage treatment works (West Yorkshire, UK)
- Method of cultivation: stored for 2 days before use; maintained at 20 ± 2°C and fed daily with synthetic sewage concentrate at a rate of 50 mL/L.
- Initial biomass concentration: 4.59 g/L (suspended solids concentration determined gravimetrically following homogenisation)
- Preparation of inoculum for exposure: adjusted with dechlorinated tap water to a nominal range of 3 ± 0.3 g/L. The pH was determined to be 6.38, and was adjusted to 7.12 with sodium hydroxide (NaOH).
- Pretreatment: none.
- Test initiation: Test vessels were inoculated in three batches of five at 30 minute intervals. Each prepared batch consisted of one control vessel and either four test substance vessels or one test substance and three reference substance vessels, as appropriate.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

Following oxygen consumption measurements to determine total respiration rates, a nitrification inhibitor (N-allylthiourea, ATU), was added as an aqueous stock solution (2.32 g/L) to the prepared blank control, test substance and reference substance vessels to give a final concentration of ca. 11.6 mg ATU/L. Oxygen consumption in the absence of nitrification was measured at least 10 minutes after amendment with ATU to assess heterotrophic respiration. Nitrification respiration was calculated as the difference between total respiration and heterotrophic respiration.

Hardness:

Not specified

Test temperature:

20 +/- 2°C

pH:

7.07 to 7.87

Dissolved oxygen:

Aerated; see tabulated oxygen consumption data

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal test concentrations:
- test substance: 1, 10, 100 and 1000 mg/L
- reference substance: 0.1, 2.0 and 40 mg/L

Details on test conditions:

The study was conducted as a combined range finding / limit test. A definitive test was not conducted in this study because as no significant inhibition was observed for total, nitrification and heterotrophic respiration in the presence of test substance during the combined range finding / limit test.

TEST SYSTEM
- Test vessel: glass flasks (containing a total volume of 250 mL)

- No. of vessels per concentration (replicates):
- test substance: 1, 10 and 100 mg/L (duplicate) and 1000 mg/L (triplicate)
- reference substance: 0.1, 2.0 and 40 mg/L (single vessel for each)
- No. of vessels per control (replicates): three; duplicate measurements were taken for each replicate control vessel, one at the start and another at the end of each run, to bracket each run
- No. of vessels per vehicle control (replicates): not applicable
- No. of vessels per abiotic control (replicates): not applicable

- Aeration: yes
- Nutrients provided for bacteria: pre-made synthetic sewage concentrate (SI093) purchased from Strathkelvin Instruments prepared in water (250 mL)
- Nitrification inhibitor used: N-allylthiourea (ATU)
- Biomass loading rate: Following inoculation, each vessel contained a nominal suspended solids concentration of ca.1.5 g/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: mains tap water
- Particulate matter: not specified

OTHER TEST CONDITIONS
- Adjustment of pH: see above.
- Photoperiod: not specified
- Light intensity: not specified
- Details on termination of incubation: At the end of the 3 hour incubation period, a portion (20 mL) of each test preparation was transferred to an appropriate sample tube containing a PTFE stirrer. The dissolved oxygen (DO) electrode was sealed in the neck of the flask, ensuring air was completely excluded. The flask contents were stirred at a constant rate during DO measurements. Oxygen consumption was measured over a period of up to 10 minutes.

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

Basis for effect:

other: inhibition of total, heterotrophic and nitrification respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Basis for effect:

other: inhibition of total, heterotrophic and nitrification respiration

Details on results:

Dissolved oxygen measurements were plotted over time. Respiration rates (mg/L/h) were determined from the gradient of the linear portion of the graph between 2.0 mg O2/L and 7.0 mg O2/L, where possible. In some cases, however, it was necessary to use readings below 2.0 mg O2/L; these were considered acceptable if the data used were with the linear portion of the curve and the rate remained consistent over the lower boundary. The graphical data and calculated respiration rates were generated using Strathkelvin Strathtox software.

Respiration rates and percent inhibition for the test substance are summarized in Table 1.

The blank control respiration rate was ≥ 20 mg O2/g/h. The coefficient of variation of the blank control respiration rates were ≤ 30%.

Inhibition of nitrification respiration (21.7%) was noted at the 10 mg/L treatment when compared to the control. This inhibition was not statistically significant (p value = 0.467). This inhibition was attributed to the very short linear gradient used to determine the respiration rate (only 52 seconds of data) and natural variation in the test medium, and therefore was not considered to be biologically significant.

The EC50 values for total, heterotrophic and nitrification respiration were concluded to be greater than 1000 mg/L. The no observed effect concentrations (NOEC) for total, heterotrophic and nitrification respiration were therefore determined to be 1000 mg/L.

Results with reference substance (positive control):

Respiration rates and percent inhibition for the reference substance are summarized in Table 2. The 3-hour EC50 values for the reference item, 3,5-dichlorophenol, were determined to be 2.35 mg/L (total respiration), 1.12 mg/L (nitrification respiration) and 7.08 mg/L (heterotrophic respiration).

Reported statistics and error estimates:

Statistical analysis was performed using the Comprehensive Environmental Toxicity Information System program (CETIS, v 1.8.6.8).

As less than 50% inhibition was observed in the presence of test substance, the EC50 for total, heterotrophic and nitrification respiration could not be determined and is therefore concluded to be greater than the highest concentration tested. The EC50 for the reference substance was based on a statistical analysis (linear interpolation) of concentration versus effect in total respiration and nitrification respiration.

Linear interpolation analysis was performed to estimate total, heterotrophic and nitrification respiration EC10, EC20 and EC50 values for the reference substance and test substance, where appropriate. A parametric-control versus treatment test (Dunnett multiple comparison test) was performed to estimate total, heterotrophic and nitrification respiration no observed effect concentrations (NOEC) for the test substance.

Table1: Respiration Rates and Percent Inhibition for the Test Substance

Test Substance Nominal Concentration (mg/L)	Rep	Total Respiration Rate (mg/L/h)	Heterotrophic Respiration Rate* (mg/L/h)	Nitrification Respiration Rate**  (mg/L/h)	Total Respiration Inhibition (%)	Heterotrophic Respiration Inhibition (%)	Nitrification Respiration Inhibition (%)
Control	A	72.2	39.9	32.3	-	-	-
	A	70.4	36.6	33.8
	B	51.5	27.6	23.9
	B	47.0	25.6	21.4
	C	51.7	28.1	23.6
	C	46.1	25.6	20.5
	Mean	56	31	26
1	A	69.9	40.2	29.7	-8.3	-11.6	-4.4
	B	52.4	28.0	24.4
	Mean	61	34	27
10	A	60.5***	42.0	18.5	2.4	-14.0	21.7
	B	49.8	27.7	22.1
	Mean	55	35	20
100	A	72.6***	37.9	34.7	-1.7	0.9	-4.8
	B	42.3	22.7	19.6
	Mean	57	30	27
1000	A	76.7****	37.2	39.5	-1.7	2.1	-6.1
	B	47.0	26.6	20.4
	A	48.6	26.0	22.6
	Mean	57	30	28
Negative values indicate stimulated respiration.
Rep: Replicate vessel
- Not Applicable
*  Respiration in the presence of a nitrification inhibitor, N-allylthiourea (11.6 mg ATU/L).
** Calculated as the difference of total respiration rate and heterotrophic respiration rate.
*** The dissolved oxygen level for replicate A at the start of the total respiration measurement was low (ca. 2 mg/L) and therefore, it was necessary to calculate the total respiration rate on a short linear gradient below 2 mg/L.
**** The dissolved oxygen level for 1000 mg/L replicate A at the start of the total respiration measurement was low (ca. 2.5 mg/L) and therefore, it was necessary to calculate the total respiration rate on a linear gradient that extended below 2 mg/L. This was not considered to have an impact on the results as the coefficient of variation of the three total respiration rates for this treatment group was 29.1 %, which is considered acceptable variation based on the criterion of <30% for the control replicates, and the total respiration rate was similar to other rates in that batch run. The respiration rate for 1000 mg/L replicate A was notably higher than the other replicates vessels (B and C), but it was consistent with the total respiration rates for the control replicate (A) in this run.

 

Table 2: Respiration Rates and Percent Inhibition for the Reference Substance

3,5-DCP Nominal Concentration (mg/L)	Total Respiration Rate (mg/L/h)	Heterotrophic Respiration Rate* (mg/L/h)	Nitrification Respiration Rate** (mg/L/h)	Total Respiration Inhibition (%)	Heterotrophic Respiration Inhibition (%)	Nitrification Respiration Inhibition (%)
0.1	52.5	28.6	23.9	7.1	6.4	7.8
2.0	29.3	22.1	7.2	48.1	27.7	72.2
40	4.4	4.1	0.3	92.2	86.6	98.8
- Not Applicable
* Respiration in the presence of a nitrification inhibitor, N-allylthiourea (11.6 mg ATU/L).
** Calculated as the difference of total respiration rate and heterotrophic respiration rate.

Validity criteria fulfilled:

yes

Conclusions:

No statistically significant effects were observed on respiration of activated sludge at the maximum limit concentration of Molybdenum trioxide, reaction products with bis[O,O-bis(2-ethylhexyl)] hydrogen dithiophosphate (oil free), following a 3-hour exposure. The EC50 values for total, heterotrophic and nitrification respiration were concluded to be greater than 1000 mg/L. The no observed effect concentrations (NOEC) for total, heterotrophic and nitrification respiration were therefore determined to be 1000 mg/L.

The validity criteria were met in this the range finding / limit test.

Executive summary:

The study was conducted to determine the no observable effect concentration (NOEC) and, where possible, the effect concentration for an x% effect (e.g. EC10, EC20, EC50) for activated sludge micro-organisms exposed to the test substance in accordance with OECD Test Guideline 209.

The activated sludge inoculum was collected from the sludge return line at Burley Menston Sewage Treatment Works (West Yorkshire, U.K.), which has a predominantly domestic catchment. The point of collection was selected to ensure that the activated sludge sample was relatively free of exogenous material.

A combined range finding / limit test, employing nominal test concentrations of 1, 10, 100 and 1000 mg/L, was undertaken to determine the appropriate concentration levels for a definitive test. A definitive test was not conducted in this study because no statistically significant inhibition was observed for total, nitrification and heterotrophic respiration during the combined range finding / limit test following a 3-hour exposure to the test substance. Inhibition (21.7%) of nitrification respiration was noted at the 10 mg/L treatment group when compared to the control. This inhibition was attributed to the very short linear gradient used to determine the respiration rate (only 52 seconds of data) and natural variation in the test medium, and therefore was not considered to be biologically significant.

The EC50 values for total, heterotrophic and nitrification respiration were concluded to be greater than 1000 mg/L. The no observed effect concentrations (NOEC) for total, heterotrophic and nitrification respiration were therefore determined to be 1000 mg/L.

The 3-hour EC50 values for the reference item, 3, 5-dichlorophenol, were determined to be 2.35 mg/L (total respiration), 1.12 mg/L (nitrification respiration) and 7.08 mg/L (heterotrophic

respiration).

The validity criteria were met in this range finding / limit test and the data are therefore considered valid.

Description of key information

In an OECD 209 study, conducted according to GLP, no significant inhibition oftotal, heterotrophic or nitrification respirationwas observed following a 3-hour exposure of activated sewage sludge to the test substance, resulting in an EC50 value of greater than 1000 mg/L.  The No Observed Effect Concentration (NOEC) following the 3-hour exposure was 1000 mg/L (Smithers Viscient (ESG) Ltd, 2018).

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L