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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Jan - 13 Apr 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

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:
The test item was weighed in ELENDT M4 dilution water followed by shaking on a shaking machine for 24 h being necessary to dissolve the test item sufficiently in the test medium. Thereafter the suspensions were passed over glass fibre filter and membrane filter in order to separate the non dissolved fraction from the dissolved fraction. The clear eluates were used directly without any further dilution steps.
- Controls: Yes

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: STRAUS, clone 5
- Source: Originally derived from the lnstitut National de Recherche Chimique Appliquee, France in 1978
- Age of parental stock (mean and range, SD): Not older than 24 h
- Feeding during test: No

ACCLIMATION
- Acclimation period: Cultivation of the daphnia is performed in a way that the animals are transferred in new test medium at an interval of 1 to 3 days.
- Acclimation conditions (same as test or not): Same
- Type and amount of food: Desmodesmus subspicatus- and/or Chlorella-cells
- Feeding frequency: An interval of 1 to 3 days

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Daphnias used for the test are not older than 24 h. Therefore, in every case, daphnia are transferred into fresh medium one day before starting of the daphnia test in order to get daphnia for the test younger than 24 h.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20.7 - 20.9 °C (Control)
20.8 - 20.9 °C (Test item concentrations)

pH:

8.89 - 9.06 (Control)
8.95 - 9.22 (Test item concentrations)

Dissolved oxygen:

7.8 - 8.2 mg O2/L (Control)
7.4 - 8.2 mg O2/L (Test item concentrations)

Nominal and measured concentrations:

Control, 11.1, 22.2, 44.4, 88.8 and 111.1 mg test item/L (nominal)
Control, 9.99, 20.0, 40.0, 88.8 and 100 mg active substance/L (nominal)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: Open
- Material, size, headspace, fill volume: 100 mL glass carboys
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt medium M4 were used to prepare the test solutions.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Test start and test end

OTHER TEST CONDITIONS
- Photoperiod: 6 h of light and 8 h of darkness.

EFFECT PARAMETERS MEASURED: Mobility of the daphnia; examined by the determination of the swimming behavior of the daphnids after gentle agitation.

VEHICLE CONTROL PERFORMED: yes/no

RANGE-FINDING STUDY
- Test concentrations: 1.0; 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Based on the results of the screening test the test item concentration range of the main test was performed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

80.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

22.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

44.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

12.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: EL20

Effect conc.:

24.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

72.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

11.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: EL20

Effect conc.:

21.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: no
- Mortality of control: no

Results with reference substance (positive control):

The reference substance was tested twice a year. The value of the EC50 (24 h) was at 0.95 mg/L potassium dichromate. This value is in accordance with the recommendations of 0.6 to 2.1 mg/L K2Cr2O7 given in OECD Guideline 202 (Daphnia sp., Acute Immobilisation Test)

Reported statistics and error estimates:

Statistical evalaution was done using the commercial computer program ToxRat Professional Version 3.2.1

Biological Results:

Table 1: Ratio of immobilised Daphnids

Nominal Concentration of the test item (mg a.i./L)	Ratio immobilised Daphnia [%]
	24 h	48 h
Control	0 [0/20] 1)	0 [0/20] 1)
9.99	0 [0/20] 1)	0 [0/20] 1)
20.0	0 [0/20] 1)	5 [1/20] 1)
40.0	65 [13/20] 1)	75 [15/20] 1)
79.9	20 [4/20] 1)	95 [19/20] 1)
100.0	30 [6/20] 1)	100 [20/20] 1)

1) Numbers in brackets give the absolute numbers of immobilized animals at each concentration.

 Table 2: Endpoints based on Test item concentration

	Basis: Nominal Concentrations [mg test item/L] ("WAF")
	t24hr	t48hr
NOEL	22.2	22.2
LOEL	44.4	44.4
EL10	18.9	12.8
EL20	40.3	24.1
EL50	171.1	80.8

 Table 3: Endpoints based on active ingredient

	Basis: Nominal Concentrations of Active Substance ("WAF") [mg Zinc, bis(O,O-bis(1-methylethyl) phosphorodithioato- kS)bis(cyclohexanamine)-,(T-4)- /L]
	t24hr	48hr
NOEL	20.0	20.0
LOEL	40.0	40.0
EL10	17.0	11.5
EL20	36.3	21.7
EL50	154.0	72.7

   

Analtical Results:

As a conclusion of the analytical part of this study, it can be stated that the test item concentrations remained sufficiently stable during incubation of 48 h. As a result of the supporting analyse it can be stated that the results of the biological part should be based on the nominal concentrations applied because the recoveries of the nominal concentrations were all >80%.

Formula of the active Substance: C24H5 4N20 4P2S2Zn

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%

  

Table 4: Basis P 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 t0 t0 t0 t0 t0	Control 10 20 40 80 100	0 1.547 2.952 3.559 8.959 9.929	- 15.5 14.8 8.9 11.2 9.9	- - - - - -

Time	nom. Conc. of active Substance [mg/Ll	Basis P
		mg Test ltem/L	% Nominal Conc.	Recovery % of t0
t48h	Control	0	-	-
t48h	10	1.234	12.3	79.7
t48h	20	2.750	13.8	93.2
t48h	40	3.246	8.1	91.2
t48h	80	8.686	10.9	97.0
t48h	100	9.353	9.4	94.2

 Table 5: Basis S

Time	nom. Conc. of active Substance [mg/L]	Basis S
		mg Test ltem/L	% Nominal Cone.	Recovery % of 10
t0 t0 t0 t0 t0 t0	Control 10 20 40 80 100	0 0.781 3.613 8.398 21.777 24.023	- 7.8 18.1 21.0 27.2 24.0	- - - - - -
t48h t48h t48h t48h t48h t48h	Control 10 20 40 80 100	0 3.906 7.324 8.301 22.070 20.996	- 39.1 36.6 20.8 27.6 21.0	- 500.0 202.7 98.8 101.3 87.4

 Table 6: Basis Zn

Time	nom. Conc. of active Substance [mg/L]	Basis Zn			Mean Recoveries, % of 10, total [Basis P, S, Zn]
		mg Test ltem/L	% Nominal Conc.	Recovery% of t0
t0 t0 t0 t0 t0 t0	Control 10 20 40 80 100	0 0.412 0.460 0.546 0.584 0.718	- 4.1 2.3 1.4 0.7 0.7	- - - - - -	- - - - - -
t48h	Control	0	-	-	-
t48h	10	0.632	6.3	153.5	244.4
t48h	20	0.766	3.8	166.7	154.2
t48h	40	0.843	2.1	154.4	114.8
t48h	80	1.561	2.0	267.2	155.2
t48h	100	1.236	1.2	172.0	117.9

Table 7: Validity criteria for OECD 202.

Criterion from the guideline	Outcome	Validity criterion fulfilled
In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.	No mortality was observed	Yes
The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.	oxygen concentrations was above 60% of the saturated value (ca 5 mg O2/L)	Yes

 

Validity criteria fulfilled:

yes

Conclusions:

The EL50 was determined to be at 80.8 mg/L after 48 h and the NOEL was observed to be at 22.2 mg/L .

Description of key information

EL50 (48 h) = 80.8 mg test item/L (Daphnia magna, nominal, OECD 202, key study)

EL50 (48 h) = 72.7 mg active ingredient/L (Daphnia magna, nominal, OECD 202, key study)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

72.7 mg/L

Additional information

One experimental sutdy on the short-term toxicity of the test item to the freshwater species Daphnia magna is available (Lebertz, 2017b). The study was performed according to OECD guideline 202. The nominal concentrations were verified by analytical monitoring. All studies were performed according to GLP. Due to the moderate water solubility the test item was weighed in test medium followed by shaking on a shaking machine for 24 h and passed over glass fibre filter and membrane filter. The clear eluates were used directly without any further dilution steps. Daphnia magna was exposed over a period of 48 h without feeding in a static system to nominal concentrations of 11.1, 22.2, 44.4, 88.8 and 111.1 mg test item/L (nominal). Based on the active ingredient D. magna was exposed to the concentrations of 9.99, 20.0, 40.0, 88.8 and 100 mg active substance/L (nominal). Under the conditions used for the test, there was an immobilisation of D.magna at concentrations ≥22 mg test item/L representing 20 mg/L of active substance/L. As a conclusion of the analytical part of this study, it can be stated that the concentrations of the test item remained sufficiently stable during incubation of 48 h. As a result of the supporting analyse it can be stated that the results of the biological part should be based on the nominal concentrations applied because the recoveries of the nominal concentrations were all >80%. The EL50 was determined to be at 80.8 mg test item/L after 48 h and the NOEL was observed to be at 22.2 mg test item/L. Based on the active ingredient the EL50 was determined to be at 72.7 mg a.i./L and the NOEL was observed to be at 20.0 mg a.i./L.