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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 August 2017 to ****

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

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:

yes

Remarks:

Given that the test substance is a multi-constituent substance and analytical monitoring was limited to the dithiophosphate fraction (without molybdenum) of the test substance, the results were based on nominal loading rates.

Details on sampling:

- Concentrations: Control, 0.10, 0.32, 1.0, 3.2, 10 and 100 mg/L loading rate WAF
- Sampling method: Aqueous samples (10 mL) of freshly prepared test media (0 hour) and pooled expired test media (72 hours) were collected in duplicate, mixed with acetonitrile, then diluted further with medium:acetonitrile (1:1, v/v) as required to bring the response within the calibration range.
- Sample storage conditions before analysis: Not applicable

Vehicle:

no

Details on test solutions:

The substance is a chemical substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB) with low water solubility and as such falls into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). Based on the recommendations of this guideline, the test solutions were prepared as Water Accommodated Fractions (WAFs).
At the start of the test, the test substance was weighed onto a glass slides and placed in flasks containing 1000 mL of OECD medium. The preparations were stirred for ca 23 hours at a rate such that a slight dimple was formed at the surface. After the stirring period, the preparations were allowed to settle for ca 1 hour, after which the aqueous phase was removed by careful mid-depth siphoning to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF. A control group was prepared in the same manner without the addition of test substance.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Oban, UK
- Age of inoculum (at test initiation): Concentrated, axenic liquid slope cultures stored under refrigeration (2-8 °C) for up to 8 months.
- Method of cultivation: Prior to testing, duplicate starter cultures were prepared and incubated under test conditions to obtain sufficient algal cells in exponential growth and to achieve a starting algae cell density of 1 x 10^4 cells/mL.

Test type:

static

Water media type:

other: OECD medium

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.3 – 23.6 °C

pH:

7.73 – 8.97

Nominal and measured concentrations:

Nominal concentrations: Control, 0.10, 0.32, 1.0, 3.2, 10 and 100 mg/L loading rate WAF
Measured concentrations in freshly prepared samples (0 hour) ranged from less than the limit of quantification (LOQ; 0.01 mg/L) to 7.81* mg/L, and at 72 hours showed measured concentrations ranging from
* This 100 mg/L LR WAF was observed to be cloudy indicating that undissolved test substance may have been present. This was considered not to have affected the integrity of the test given that no inhibition of growth was observed at this loading rate or in the 10 mg/L LR WAF that showed a measured concentration closer to the expected solubility value.

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile, autoclaved glass 250 mL Erlenmeyer (conical) flasks
- Fill volume: 100 mL
- Aeration: none; shaken continuously (100 rpm) to keep algae in suspension and facilitate transfer
of CO2.
- Renewal rate of test solution: not applicable; static
- Initial cell density: 1 x 10^4 cells/mL
- Control end cell density: 51.6 x 10^4 cells/mL
- No. of vessels per concentration (replicates): Three test vessels were prepared for each of the 0.10, 0.32, 1.0, 3.2 and 10 mg/L loading rate WAFs. Six test vessels were prepared for 100 mg/L loading rate WAF. This design was employed to ensure that if no effects were observed, the requirements for a Limit Test were satisfied whilst also allowing for the investigation of possible low level toxicity.
- No. of vessels per control (replicates): Six
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes; OECD medium


OTHER TEST CONDITIONS
- Intervals of water quality measurement: start and end of test; temperature also was recorded continuously using minimum/maximum thermometer.
- Sterile test conditions: yes
- Adjustment of pH: none reported.
- Photoperiod: constant light
- Light intensity and quality: 5680 – 6040 lux (0 hour); 5090 – 5830 lux (72 hours); under florescent lights, emitting white light across the visible portion of the spectrum (400 - 700 nm).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: particle counter (Z2 Coulter Counter); see Table 2.
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Range finding study: 0 (control), 1.0, 10 and 100 mg/L loading rate WAF (prepared in duplicate)
- Results used to determine the conditions for the definitive study: see Table 1
- Definitive test concentrations: 0 (control), 0.10, 0.32, 1.0, 3.2, 10 and 100 mg/L loading rate WAF
- Spacing factor for test concentrations: 3.2 (with an additional dose a limit concentration)

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Basis for effect:

other: (for growth rate and yield)

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

other:

Remarks:

(growth rate and yield)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none reported
- Unusual cell shape: none reported
- Colour differences: none reported
- Flocculation: none reported
- Adherence to test vessels: none reported
- Aggregation of algal cells: none reported
- Any stimulation of growth found in any treatment: yes
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none specified
- Effect concentrations exceeding solubility of substance in test medium: yes

Results with reference substance (positive control):

A positive control study was conducted to determine the effects of the reference substance, potassium dichromate, on algal growth by exposing the green alga, Pseudokirchneriella subcapitata, at the exponential growth phase to the reference substance for 72 hours. The 72-hour EC50 values for yield, area under the growth curve and growth rate were determined to be 0.599, 0.604 and 0.875 mg/L, respectively. The 72-hour NOEC value for yield, area under the growth curve and growth rate were determined to be 0.32, <0.1 and 0.32 mg/L, respectively.

These results were within the expected toxicity range (growth rate EC50: 0.38 - 2.60 mg/L) for this reference substance and all validity criteria were met; the test is therefore considered valid.

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v 1.8.6.8. The final yield and average specific growth rate (µ) over specified time intervals were analyzed using Bonferroni Adjusted t-test to determine the no observed effect effect loading rate (NOEL). Linear interpolation analysis was performed in order to estimate EyC50 (yield) and ErC50 (growth rate).

Table 1: Summary of results from the range-finding test

Nominal	Specific growth rate		Yield
loading rate (mg/L)	0 to 72 hours (cells/mL)	% Inhibition	0 to 72 hours (cells/mL)	% Inhibition
Control	6.617	NA	6.617E-02	NA
1.0	5.840	12	5.840	12
10	6.583	1	6.583	1
100	6.369	4	6.369	4

 NA – Not applicable

 

Samples were collected for chemical analysis from the range-finding test; the analytical batch failed, however, due to high QC recovery values. Media trials were therefore conducted to investigate the amount of test substance present in a WAF prepared from an initial 100 mg/L loading rate. The WAFs were prepared using a slow stir for a period of 23 hours followed by a settlement period of 1 hour. The WAFs were removed by careful mid-depth siphon and analyzed based on the solubledithiophosphate fraction (without molybdenum).The WAF from one of the preparations (100 mg/L) was slightly cloudy, indicating that undissolved test substance may have been present. The results of the media preparation trial indicated that the function solubility of thedithiophosphate fractionof the test substance (without molybdenum)in OECD medium was approximately 1 mg/L.

 

The freshly prepared test media at 0 hours in the definitive test appeared as colourless solutions with the exception of the 100 mg/L loading rate WAF, which was observed to be cloudy.The appearance of the test media following the 72-hour definitive exposure period was described as light green with a homogenous, hazy dispersion of algae cells.

 

 Table 2: Percent inhibition of growth rate of Pseudokirchneriella subcapitata

Nominal loading rate	Average specific growth rate, “µ” (cells x 10^-2/mL)				Inhibition of average specific growth  rates relative to the control (%)
(mg/L)	0-24 hours	24-48 hours	48-72 hours	0-72 hours	0-24 hours	24-48 hours	48-72 hours	0-72 hours
Control	4.31	5.99	6.10	5.47	-	-	-	-
0.10	4.84	5.89	6.52	5.75	-12	2	-7	-5
0.32	4.18	7.69	6.63	6.17	3	-28	-9	-13
1.0	4.46	6.55	5.78	5.60	-3	-9	5	-2
3.2	4.81	7.09	6.72	6.21	-12	-18	-10	-14
10	5.23	7.31	6.68	6.50	-28	-22	-10	-19
100	6.42	5.09	6.43	5.98	-49	15	-5	-9

 - Not applicable

Negative percentage inhibition values relative to the control indicate growth stimulation and are not considered an adverse effect.

 

 Table 3: Percent inhibition of yield of Pseudokirchneriella subcapitata

Nominal loading rate (mg/L)	Initial cell density (cells/mL)	Yield at 72 hours * (cells/mL)	Mean Final Yield per treatment (cells/mL)	% Inhibition in Final Yield relative to control treatment
Control	1E04	474720 564500 562320 557320 419840 455300	50.6E04	-
0.10	1E04	533940 670820 656420	62.0E04	-23
0.32	1E04	685180 923600 928580	84.6E04	-67
1.0	1E04	528940 485280 655760	55.7E04	-10
3.2	1E04	733360 825400 1057540	87.2E04	-72
10	1E04	1081240 1049080 1082380	107E04	-112
100	1E04	708320 814160 765580 759060 741880 612740	73.4E04	-45

 * Final yield (cells/mL) = cell density (cells/mL) at 72 hours – initial cell density (cells/mL)

- Not applicable

Negative percentage inhibition values relative to the control indicate growth stimulation and are not considered an adverse effect.

Validity criteria fulfilled:

yes

Remarks:

The control cell densities increased by a factor of 51. The mean coefficient of variation (CV) for section-by-section specific growth rate in the control cultures was 19.5% and the overall CV for specific growth rate was 3.19%.

Conclusions:

Based on nominal loading rates, the 72-hour EyL50 and the 72-hour ErL50 values were determined to be >100 mg/L loading rate WAF. The corresponding No Observed Effect Loading rate (NOEL) values for yield and specific growth rate after 72 hours were determined to be 100 mg/L loading rate WAF. The test showed no toxicity at the limit of solubility of the test substance in the test media.

Executive summary:

The study was conducted in accordance with the requirements of OECD Chemicals Testing Guideline No. 201 to assess the effects of the test substance on the growth of the freshwater green alga, Pseudokirchneriella subcapitata, during a 72-hour growth inhibition toxicity test. Test solutions were prepared as Water Accommodated Fractions (WAFs) based on the recommendations of OECD Series on Testing and Assessment, No. 23.

Based on the results of a range finding test and media preparation trials, the definitive test was conducted under static conditions at nominal loading rates of 0.10, 0.32, 1.0, 3.2, 10 (three replicates each) with an additional limit concentration of 100 mg/L (six replicates) and a concurrent control group (six replicates). Test vessels (250 mL conical flasks) were prepared containing 100 mL of the appropriate test or control medium. Each test vessel was inoculated with 1 x 10^4 algae cells/mL and incubated at 21 - 24°C with constant lighting (5090 – 6040 lux) for 72 hours. Cell counts and water quality measurements were performed at 24-hour intervals.

Samples of test media were analysed at 0 (new media) and 72 hours (expired test media) using high performance liquid chromatography (HPLC) with time of flight mass spectrometry (TOF) based on quantitation of the dithiophosphate fraction (without molybdenum) of the test substance. Measured concentrations at 0 hours ranged from less than the limit of quantification (LOQ; 0.01 mg/L) to 7.81 mg/L and at 72 hours ranged from less than the limit of quantification to 2.64 mg/L. Given that the test substance is a multi-constituent substance and analytical monitoring was limited to the dithiophosphate fraction (without molybdenum) of the test substance.

The 0-72 hour ErL50 (growth rate) and the 72-hour EyL50 (yield) values were determined to be >100 mg/L loading rate WAF. The corresponding No Observed Effect Loading rate (NOEL) values for yield and specific growth rate after 72 hours were determined to be 100 mg/L loading rate WAF. The test showed no toxicity at the limit of solubility of the test substance in the test media. All validity criteria were met and the test was therefore considered valid.

Description of key information

In an OECD 201 study, conducted according to GLP, the effect of the test substance on the growth of Pseudokirchneriella subcapitata was investigated and the 72 hour EL50 value based on growth rate was determined to be greater than 100 mg/L loading rate WAF and the No Observed Effect Loading rate (NOEL) was determined to be 100 mg/L loading rate WAF (Smithers Viscient (ESG) Ltd, ****). 

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L