Bis(O,O-diisopropyl dithiophosphate)bis(cyclohexylamine) 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:

10 Jan - 16 Mar 2017

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

Deviations:

yes

Remarks:

The concentration of NaHCO3 was increased to be twice of that indicated in the Guideline 201. This cocnentration has been found to be optimal.

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and all test item concentrations
- Sampling method: samples were taken at test start and test end
- Sample storage conditions before analysis: samples were stored at 2 to 8 °C until they were analysed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Differential loading:
Separate solutions were made for each test concentration in ultrapure water. Aliquots of these solutions were made up with ,,intermediate dilution" and algal inoculum (or diluted ,,intermediate dilution") to give 50 mL of final volume.Suspensions of the test item were individually prepared for each test concentration in ultrapure water and shaken on a shaking machine at approx 130 rpm for 24 hrs at RT and filtrating through glass fibre filter. The eluates were used without further dilution steps (detailed information are summarized within the section "Any other information on material and methods")
- Controls: yes
- Others: As reagents' blank without algal inoculation three test solutions were prepared as described above containing a 1:10 diluted intermediate dilution of the mineral nutrients stock solution instead of the algal pre-culture.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: 86.81
- Source: Sammlung von Algenkulturen, Pflanzenphysiologisches lnstitut der Universität Göttingen
- Age of inoculum (at test initiation): 72 h (pre-culture)

ACCLIMATION
- Acclimation period: 3 d
- Culturing media and conditions: same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 - 26 °C

pH:

8.12 - 9.18 (Control)
8.09 - 9.36 (Test item concentration)

Nominal and measured concentrations:

Control, 11.1, 22.2, 44.4, 88.9 and 177.8 mg test item/L (nominal)
Control, 10, 20, 40, 80 and 160 mg active ingredient/L (nominal)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: 50 mL glass cylinders, 50 mL volume;
- Initial cells density: 3 x 1000
- Control end cells density: 3.3 x 100000
- No. of vessels per concentration (replicates): 7
- No. of vessels per control (replicates): 7

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ultra pure water
- Intervals of water quality measurement: test start and test end

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: constant illumination
- Light intensity and quality: ≥120 µE/m²s (ca 8000 lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: photometrical measurement (spectrophotometer at 578 nm); cell numbers were calculated using a calibration curve of cell numbers versus extinction values (578 nm)
- Chlorophyll measurement:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

95.43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

44.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

85.86 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities: no

Reported statistics and error estimates:

Estimation of the inhibition values for yield and the growth rates of the algae as well as the statistical calculations were conducted according to the Guideline using the commercial computer program ToxRatPro Version 3.2.1.

Results of Analytics:

Formula of the active Substance: C24H54N204P2S2Zn

According to this formula the contents of the different elements in the molecule are distributed as C: 46.04%; H: 8.69%; N: 4.7%; O: 10.22 %; P: 9.89%; S: 10.24 %; Zn: 10.44%

Amount of P, S and Zn in the treatments measured:

Time	Concentration of the Test Item, active Substance [mg/L]	mg/L
		P	S	Zn
t0	Control	0.486	2.128	0.006
t0	10.01	0.599	2.421	0.084
t0	19.94	0.720	2.651	0.205
t0	40.00	0.981	3.196	0.265
t0	79.99	1.481	4.315	0.299
t0	159.98	3.301	8.120	0.438
t72h	Control	0.410	2.040	0.004
t72h	10.01	0.584	2.498	0.102
t72h	19.94	0.740	2.929	0.224
t72h t72h t72h	40.00 79.99 159.98	1.044 1.771 3.270	3.325 4.880 8.100	0.298 0.298 0.445

Accordingly, the following concentrations of active substance are calculated:

Time	nom. conc. of active substance [mg/L]	Measured conc. of active substance
		Basis P	Basis S	Basis Zn
		mg Test ltem/L	mg Test ltem/L	mg Test ltem/L
t0	Control	0.000	0.000	0.000
t0 t0	10.01 19.94	1.143 2.366	2.861 5.107	0.747 1.906
t0	40.00	5.005	10.430	2.481
t0	79.99	10.061	21.357	2.807
t0	159.98	28.463	58.516	4.138
t72h t72h t72h t72h t72h t72h	Control 10.01 19.94 40.00 79.99 159.98	0.000 1.759 3.337 6.411 13.761 28.918	0.000 4.473 8.682 12.549 27.734 59.180	0.000 0.939 2.107 2.816 2.816 4.224

Accordingly, the following recovery rates are calculated:

Time	nom. conc. of active substance [mg/L]	Basis P
		mg Test ltem/L	% Nominal Conc.	Recovery% of t0
t0	Control	0	-	-
t0 t0	10.01	1.143	11.4	-
t0	19.94	2.366	11.9	-
t0	40.00	5.005	12.5	-
t0	79.99	10.061	12.6	-
t0	159.98	28.463	17.8	-
t72h	Control	0	-	-
t72h	10.01	1.759	17.6	154.0
t72h	19.94	3.337	16.7	141.0
t72h	40.00	6.411	16.0	128.1
t72h	79.99	13.761	17.2	136.8
t72h	159.98	28.918	18.1	101.6

Time	nom. conc. of active substance [mg/L]	Basis S
		mg Test ltem/L	% Nominal Conc.	Recovery % of t0
t0 t0 t0 t0 t0 t0	Control 10.01 19.94 40.00 79.99 159.98	0 2.861 5.107 10.430 21.357 58.516	- 28.6 25.6 26.1 26.7 36.6	- - - - - -
t72h t72h t72h t72h t72h t72h	Control 10.01 19.94 40.00 79.99 159.98	0 4.473 8.682 12.549 27.734 59.180	- 44.7 43.5 31.4 34.7 37.0	- 156.3 170.0 120.3 129.9 101.1

Time	nom. Conc. of active substance [mg/L]	Basis Zn			Mean Recoveries, % of t 0, total [Basis P, 5, Zn]
		mg Test ltem/L	% Nominal Conc.	Recovery % of t0
t0 t0 t0 t0 t0 t0	Control 10.01 19.94 40.00 79.99 159.98	0 0.747 1.906 2.481 2.807 4.138	- 7.5 9.6 6.2 3.5 2.6	- - - - - -	- - - - - -
t72h t72h t72h t72h t72h t72h	Control 10.01 19.94 40.00 79.99 159.98	0 0.939 2.107 2.816 2.816 4.224	- 9.4 10.6 7.0 3.5 2.6	- 125.6 110.6 113.5 100.3 102.1	- 145.3 140.5 120.6 122.3 101.6

Table 1: Validity criteria for OECD 201

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	123.2	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	16.4%	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	3.0%	Yes

Validity criteria fulfilled:

yes

Description of key information

ErL50 (72h) >100 mg test item/L (Desmodesmus subspicatus, nominal, OECD 201, growth rate, key study)

ErL50 (72h) > 100 mg a.i./L (Desmodesmus subspicatus, nominal, OECD 201, growth rate, key study)

ErL10 (72 h) 95.43 mg test item/L (Desmodesmus subspicatus, nominal, OECD 201, growth rate, key study)

ErL10 (72 h) 85.86 mg a.i./L (Desmodesmus subspicatus, nominal, OECD 201, growth rate, key study)

Additional information

One experimental study is available investigating the toxicity of the test item to the freshwater green algae Desmodesmus subspicatus (Lebertz, 2017c). The study was performed according to OECD 201 and GLP with analytical monitoring of the test item concentration during the exposure period of 72 hours. The test was performed using five concentrations of "WAFs" (Water accommodated fractions) in the nominal range of 11.1 to 178.6 mg/L of the test item representing 10 to 160 mg/L of active matter (90% of the test item are representing the active matter). Suspensions of the test item were individually prepared for each test concentration in ultrapure water and shaken on a shaking machine at approximately 130 rpm for 24 h at room temperature. After the shaking procedure the suspensions were filtered through glass fibre filters. The eluates (WAFs) were used directly without any further dilution step. As a result of the supporting analyse it can be stated that minor concentrations of the test item were measured due to WAF-preparation. Nevertheless the measured concentrations remained stable during incubation and thus, the results of the biological part should be based on the nominal concentrations applied because the necessary stability of 80% could be shown. The EL50 (72h) for growth rate was determined to be > 100 mg/L based on the active ingredient as well as on the test item. The EL10 (72h) for growth rate was determined to be at 95.43 mg test item/L and based on the active ingredient it was determined to be at 85.86 mg a.i./L. The validity criteria for the study as given by the OECD Guideline 201 were all met and the study is therefore considered to be valid.