bis((bis(dodecyloxy)phosphorothioyl)thio)zinc

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:

May 17, 2018 - August 30, 2018

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)

Version / remarks:

2006 (corrected 2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Quadruplicate samples were taken from the test media of all test concentrations and the control at the start of the test (without algae).
After 24, 48 hours and at the end of the test (after 72 hours), stability samples (containing algae) were taken in quadruplicate from all test concentrations and from the control. The analytical stability samples (10 mL sample volume) at 24 and 48 hours could not be taken from the test vessels itself due to the limited test medium volume (30 mL). Therefore, for each of these sampling dates, a set of additional flasks containing the corresponding test medium (with algae) was incubated under conditions identical to the test.

For the 72-hour stability samples of the test media with algae, the contents of the respective replicates were combined prior to sampling. Immediately after sampling, nitric acid (0.7 mL 70% nitric acid per 10 mL sample volume) was added to each sample in order to stabilize the latter during the storage period. Thereafter, all samples were stored frozen (at about -20 °C). In pre-experiments for investigation of the storage stability of the samples (non-GLP), the test item proved to be stable under these storage conditions. Two of the quadruplicate test medium samples from the undiluted filtrate with the loading rate of 100 mg/L and the control were analyzed per sampling time. The remaining two samples were taken as precaution to enable additional analyses and were stored frozen (at about -20 ± 5 °C). The samples from the test dilutions 6.25 to 50 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Differential loading: 100, 50, 25 12.5, 6.25 mg/L
- Controls: test water only
- Test concentration separation factor: 2


A non-GLP pre-experiment to determine the appropriate test design (poor soluble test item; water solubility < 100 mg/L) was performed. A stirring time of 96 hours (highest amount of test item dissolved in test water) was chosen for preparation of the test media used in the range finding and the main test.

226.2 µL test item were carefully applied (pipetted) onto the surface of 2330 mL test water (loading rate of 100 mg/L). No auxiliary solvent or emulsifier was used. The resulting test medium was slowly stirred for 96 hours at room temperature and in the dark. The mixing vessel was nearly completely filled (a small headspace had to be included as the test item was floating on the water surface) and tightly sealed with glass stoppers. After this treatment the lower part of the equilibrated test medium was carefully harvested from the mixing vessel through a tap at the bottom of the vessel. The rest of the test medium with undissolved test item floating on the surface remained in the stirring vessel. This equilibrated aqueous phase with a loading rate of 100 mg/L was filtered through a membrane filter (Whatman, Type NC45, pore size 0.45 μm). As a pre-caution, the filter was pre-conditioned with 200 mL filtrate to avoid losses of dissolved test item due to adsorption on the filter material. This undiluted filtrate was used as highest test concentration and was diluted with test water to obtain the test media with the lower test concentrations. The test media were prepared just before the start of the test.
All solutions were clear with no evidence of undissolved test item.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricomutum
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): SAG, Istitute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany
- Method of cultivation: standardized conditions according to the test guidelines

ACCLIMATION
- Acclimation period: four days
- Culturing media and conditions (same as test or not): same
The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

15 mg CaCO3/L

Test temperature:

23 °C

pH:

7.4 - 8.9

Nominal and measured concentrations:

Nominal concentrations: 100, 50, 25 12.5, 6.25 mg/L

The determination of the concentration of the test item Z-112 was based on the analytical measurement of zinc, representing 7.2 % of the test item. For details on the analytically measured concentrations of the test item see "Z-112 - results acute toxicity to algae.pdf". The mean measured concentration (arithmetic mean) of an undiluted filtrate of an equilibrated test medium with a loading rate of 100 mg/L was 1185 µg/L. The zinc concentration did not decrease over the test period. The samples from the test dilutions 6.25 to 50 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test.

The reported biological results are based on the loading rates.

Details on test conditions:

TEST SYSTEM
- Test vessel: 75 mL Erlenmeyer flasks covered with a glass lid
- Fill volume: 30 mL
- Aeration: continuous shaken
- Initial cells density: 5000 cells/mL (0.85 x 10^4 fluoreszenz units)
- Control end cells density: 192 x 10^4 fluoreszenz units
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP Medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile purified water
- Culture medium different from test medium: no

MONITORING OF EXPERIMENTAL CONDITIONS
- light intensity: measured at 9 places in the experimental area at the end and the start of the test
- pH: in each treatment at the start and the end of the test
- temperature: was monitored and recorded continuously in the incubator
- appearance of the test media: visually controlled and recorded daily

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: LED light with the standard range from 73 to 76 µE s-1 m-2

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: electronic particle counter (Cell Counter CASY TT, OLS, Bremen/Germany)
- Morphology: examined visually with the aid of a microscope at the start and end of the test

TEST MEDIUM PREPARATION
- non-GLP pre-experiment to determine the appropriate test design (poor soluble test item; water solubility < 100 mg/L): stirring time of 96 hours (highest amount of test item dissolved in test water) was chosen for preparation of the test media used in the range finding and the main test

RANGE FINDING STUDY
- non-GLP range finding test
- Test concentrations: 100 mg/L, 100 mg/L (filtered trough 0.45 µm membrane filter), 10 mg/L, 1mg/L, control
- Results used to determine the conditions for the definitive study: effects could be expected at nominal test substance concentrations > 10 mg/L

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

CULTURING APPARATUS
-Details on culturing apparatus used: temperature controlled orbital shaker (Multitron-Pro, Infors HT, Bottmingen, Switzerland)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, twice a year

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

results are expressed in terms of loading rates

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

results are expressed in terms of loading rates

Details on results:

- Exponential growth in the control (for algal test): yes, increased by a factor of 224 over 72 hours
- Mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours: 27 % (Criterion: must not be higher than 35 %)
- Coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours: 0.5 % (Criterion: must not be higher than 7 %)

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the mean measured test item concentration of 1185 μg/L and the algal cells in the control. Thus, the shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.
No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

- Effect concentrations exceeding solubility of substance in test medium: none

For details on results see attached document "Z-112 - results acute toxicity to algae.pdf".

Results with reference substance (positive control):

The 72-hour EC50 for growth rate in the reference test was 1.3 mg/L (April 2018, IES Study Number 20180052) and showed that the sensitivity of the test system was within the range recommended by the guideline (72-hour EC50 for the growth rate 0.92-1.5 mg/L).

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the test item Z-112 had no toxic effects on Pseudokirchneriella subcapitata
during the 72-hour test period up to and including the loading rate of 100 mg/L.

Description of key information

Z-112 had no toxic effects on Pseudokirchneriella subcapitata up to and including a loading rate of 100 mg/L (OECD 201, EU method C.3).

Additional information

NOEC ≥ 100 mg/L loading rate 

EC₅₀ > 100 mg/L loading rate

The test results indicate that the test material is not inhibitory to growth of algae up to the limits of its water solubility and a NOEC cannot be determined.