Reaction products of disubstituted alkane, nickel, (Z/E)-substituted alkene, reaction mass of tetraalkyl-3,3’-di(propan-2-yl)-biphenyl-diyl tetrakis(dialkylphenyl) bis(phosphite) and tris(dialkylphenyl) phosphite and zinc halide

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:

2018-10-03 to 2018-11-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 23, Guidance document on aquatic toxicity testing of difficult substances and mixtures

Version / remarks:

2000

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Frequency:
At the start (t=0 h), after 24 h and the end of the test (t= 72 h).
Concentrations:
Samples for analysis were taken from all concentrations and the control, immediately before transfer to the individual replicates. Care was taken not to include any floating layer, test item film or undissolved material in separate vessels. At the end of the test, samples were taken from the approximate center of the pooled solutions of the vessels containing the algal suspensions at each concentration.
Number of samples:
Sampling consisted of single samples per treatment. Additionally, samples were taken from the additional vessels without algae.
Volume:
2 mL per sample;
Storage:
Samples were stored in a freezer (=-15°C) until analysis

Vehicle:

no

Details on test solutions:

Water Accommodated Fractions were prepared:
Individual loading rates were vigorously agitated on a magnetic stirrer for 6 days. On day 3 of stirring, pH was measured in case adjustment would be necessary (no adjustment was necessary: pH of 8.0 ± 0.3). After 6 days of stirring, the obtained mixtures were allowed to settle for 1 day in order to allow separation of phases. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning over glasswool (put into a funnel). Glasswool filtering to avoid a positive Tyndall effect was necessary only in the range-finding test. In this case, glasswool was pre-saturated with the test item beforehand. No Tyndall effect was observed in any prepared solution, and solutions were clear and colourless at the end of the preparation procedure.
Thereafter, the aqueous WAFs were used as test concentrations.
Based on preliminary results, the period of 6 days of stirring was used to ensure that
a) Concentration of the relevant transition metal stabilizes at the pH 8.0 ± 0.3 used for ecotoxicity testing to prevent any precipitation during the test;
b) Saturation of the aquatic phase with organic hydrolysis products stemming from aromatic phosphites is obtained.

Test organisms (species):

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

Details on test organisms:

Species: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

FRESH WATER ALGAE CULTURE
Stock culture:
Algae stock cultures are started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions are continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity:
60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium:
M1; according to the NPR 6505, formulated using Milli-RO water and with the following composition:
NaNO3: 500 mg/L
K2HPO4: 39.5 mg/L
MgSO4.7H2O: 75 mg/L
Na2CO3: 20 mg/L
C6H8O7.H2O: 6 mg/L
NH4NO3: 330 mg/L
CaCl2.2H2O: 35 mg/L
C6H5FeO7.xH2O: 6 mg/L
H3BO3: 2.9 mg/L
MnCl2.4H2O: 1.81 mg/L
ZnCl2: 0.11 mg/L
CuSO4.5H2O: 0.08 mg/L
(NH4)6Mo7O24.4H2O: 0.018 mg/L

Pre-culture:
2 to 4 days before the start of the test, cells from the algal stock culture are inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture is maintained under the same conditions as used in the test. The cell density is measured immediately before use.
Pre-culture medium:
M2; according to the OECD 201 Guideline (OECD medium), formulated using Milli-RO water.
Hardness (Ca+Mg): 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

Hardness (Ca+Mg): 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 +/- 1 °C

pH:

8.0 ± 0.3

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

For the initial range-finding and the final test, the following water accommodated fractions
(WAF, based on nominal loading rate) were individually prepared:
1.0 mg/L; 10 mg/L; 100 mg/L (ranage finding test);
1.0, 3.2, 10, 32, 100 mg/L (final test).
Please see IUCLID section "Any other information on results incl. tables" below for information on analytical results.

Details on test conditions:

Please see IUCLID section "Any other information on materials and methods incl. tables" below for details.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Remarks:

Based on Water Accommodated Fraction (WAF) approach

Effect conc.:

12.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

95% CI: 11.8-13.2

Duration:

72 h

Dose descriptor:

EL10

Remarks:

Based on Water Accommodated Fraction (WAF) approach

Effect conc.:

5.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

95% CI: 5.3-6.6

Duration:

72 h

Dose descriptor:

NOELR

Remarks:

Based on Water Accommodated Fraction (WAF) approach

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on biological relevance

Details on results:

See IUCLID section "Any other information on results incl. tables" for details.

Results with reference substance (positive control):

The effect concentrations obtained for potassium dichromate were in the valid range.

Reported statistics and error estimates:

See IUCLID section "Any other information on materials and methods incl. tables" for details.

These data are based on results from draft report and will be amended as soon as the final report will have been issued.

Details on Biological Data:

Range-finding test - growth rate  

Treatm. [waf mg/L]	Mean growth rate [/d]	Std. Dev.	n	%Inhibition
Control	2.010	0.0129	6
1.0	1.991	0.0264	3	0.96
10	1.186	0.0533	3	41
100	0.520	0.0725	6	74

At the highest test item concentration (WAF 100 mg/L) turbidity was observed. Based on these effect data, a full final test was performed.

Final Test

A) GROWTH RATE

Treatm. [waf mg/L]	Mean growth rate [/d]	Std. Dev.	n	%Inhibition
Control	1.776	0.0278	6
1.0	1.722	0.0299	3	3.0
3.2	1.711	0.0363	3	3.7
10	1.154	0.1295	3	35
32	0.081	0.0706	3	95
100	0.000	0.0000	3	100

B) YIELD

Treatm. [waf mg/L]	Mean	Std. Dev.	n	%Decrease
Control	205.7	17.11	6
1.0	174.6	16.07	3	15
3.2	169.0	18.63	3	18
10	32.4	11.41	3	84
32	0.3	0.26	3	100
100	0.0	0.00	3	100

For WAF preparation, no glasswool was used since none of the test solutions were hazy before siphoning. All test solutions were clear and colorless at the end of the preparation procedure. No Tyndall effect was observed in any prepared solution. At the end of preparation, pH was measured and was within the limits as described in the Study Plan (pH of 8.0 ± 0.3).

At the end of the test, all validity criteria as described in the study plan were met. A concentration-related increase of growth rate and yield inhibition was observed.

RESULTS OF STATISTICAL EVALUATION

The following effect parameters were determined using ToxRat Professional (ToxRat Solutions®, GmbH, Germany):

Parameter (mg/L)		NOELR*	NOELR^#	EL10	ECL20	EL50
Growth rate	Value	<1.0	3.2	5.9	7.6	12.4
	lower 95%-cl			5.3	7.1	11.8
	upper 95%-cl			6.6	8.2	13.2
Yield	Value	<1.0	<1.0	2.5	3.3	5.5
	lower 95%-cl			2.2	3.0	5.1
	upper 95%-cl			2.8	3.6	5.9

cl – confidence limit, *) - based on statistical significance, #)- based on biological relevance.

Details on Analytical Data:

At this time, analytical data are available for the range-finding test, only. Data on the final test will be amended as soon as they will become available.

1) Analysis of the relevant transition metal by ICP-MS

Time of sampling hours	Date of sampling	Date of analysis	Loading rate (WAF) [mg/L]	Concentration test item analysed [mg/L]	Relative to initial [%]	Transition metal concentration calculated from elemental fraction (0.8%) determined in study number 519586 [mg/L]
0	15 Oct 2018	24 Oct 2018	1	0.37		0.003
			100	23.1		0.185
24	16 Oct 2018	24 Oct 2018	1	0.34	92	0.003
			100	23.2	100	0.186
72	18 Oct 2018	24 Oct 2018	1	0.34	92	0.003
			100	23.7	103	0.190

2) Analysis of the organics fraction by LC-UV at 210 nm

Time of sampling [hours]	Date of sampling	Date ofanalysis	Loading rate WAF [mg/L]	Concentration analysed [mg/L]	Relative to initial [%]
0	15 Oct 2018	31 Oct 2018	1.0	1.86
			100	221
24	17 Oct 2018	31 Oct 2018	1.0	0.755	41
			100	15.8	7.1
72	15 Oct 2018	31 Oct 2018	1.0	0.049	2.7
			100	0.44	0.20

Validity criteria fulfilled:

yes

Conclusions:

The toxicity to freshwater green algae was tested according to OECD 201 compliant with GLP. Due to the nature of Step 2 catalyst (UVCB), according to OECD 23 water accommodated fractions
(WAF) were prepared.
Results:
NOELR (Pseudokirchneriella subcapitata; 72 h; static; growth rate) = 3.2 mg/L;
EL10 (Pseudokirchneriella subcapitata; 72 h; static; growth rate) = 5.9 mg/L (95% CI: 5.3-6.6)
EL50 (Pseudokirchneriella subcapitata; 72 h; static; growth rate) = 12.4 mg/L (95% CI: 11.8-13.2)

Executive summary:

The toxicity to freshwater green algae was tested according to OECD 201 compliant with GLP. The test organism was Pseudokirchneriella subcapitata. Due to the nature of Step 2 catalyst (UVCB), according to OECD 23 water accommodated fractions (WAF) were prepared and based on the results of a range finding tests (significant effects observed at WAF 10 and 100 mg/L) the final test was conducted at WAFs were prepared at 1.0, 3.2, 10, 32 and 100 mg/L nominal loading: individual loading rates were vigorously agitated on a magnetic stirrer for 6 days. Based on preliminary results, the period of 6 days of stirring was used to ensure that

a) Concentration of the relevant transition metal stabilizes at the pH 8.0 ± 0.3 used for ecotoxicity testing to prevent any precipitation during the test;

b) Saturation of the aquatic phase with organic hydrolysis products stemming from aromatic phosphites is obtained.

The presence of any non-dissolved test item fraction could be excluded before testing by measuring the Tyndall effect.

Analytical monitoring was performed for the relevant transition metal (ICP-MS) as well as the organic fraction (UPLC-UV at 210 nm). At WAF 1 mg/L and 100 mg/L transition metal concentration was stable over the 72 hours of the test. Step 2 catalyst concentration equivalent of 0.37 and 23.1 mg/L, respectively, was found in freshly prepared WAF (after 6 days of stirring and one day settling period). For WAF 1 mg/L, 92% of this initial concentration was recovered after 24 and 72 hours (0.34 mg/L). For WAF 100 mg/L, 100% and 103% of the initial concentration was recovered after 24 and 72 hours (23.2 / 23.7 mg/L). This is equivalent to transition metal concentrations of ca. 3 µg/L for WAF 1 mg/L and ca. 190 µg/L for WAF 100 mg/L.

With regard to the organics fraction monitored via UPLC-UV at 210 nm, initial Step 2 catalyst concentration equivalents of 1.86 mg/L and 221 mg/L were measured for WAF 1 and WAF 100 mg/L, respectively. Over the time course of the study (72 h) there was a relevant decline to 2.7 % of initial for WAF 1 mg/L and 0.2% of initial for WAF 100 mg/L. The reason for this decline is not clear. In the corresponding test for acute toxicity on Daphnia and fish an increase of absorption at 210 nm was observed. The decrease over time observed here could be either due to adsorption to algal biomass and/or further abiotic transformations (hydrolysis / oxidation) to molecular species with different chromatographic elution time / different absorption maxima during the test. The results confirm in any case that WAF preparation succeeded in a relevant organic fraction present at least at the beginning of the test.

The following results were obtained in the final test (based on growth rate inhibition):

NOELR (Pseudokirchneriella subcapitata; 72 h; static; growth rate) = 3.2 mg/L;

EL10 (Pseudokirchneriella subcapitata; 72 h; static; growth rate) = 5.9 mg/L (95% CI: 5.3-6.6)

EL50 (Pseudokirchneriella subcapitata; 72 h; static; growth rate) = 12.4 mg/L (95% CI: 11.8-13.2)

Description of key information

The toxicity to freshwater green algae was tested according to OECD 201 compliant with GLP. Due to the nature of Step 2 catalyst (UVCB), according to OECD 23 water accommodated fractions (WAF) were prepared.

Results:

NOELR (Pseudokirchneriella subcapitata; 72 h; static; growth rate) = 3.2 mg/L;

EL10 (Pseudokirchneriella subcapitata; 72 h; static; growth rate) = 5.9 mg/L (95% CI: 5.3-6.6)

EL50 (Pseudokirchneriella subcapitata; 72 h; static; growth rate) = 12.4 mg/L (95% CI: 11.8-13.2)

Key value for chemical safety assessment

EC50 for freshwater algae:

12.4 mg/L

EC10 or NOEC for freshwater algae:

5.9 mg/L

Additional information

The toxicity to freshwater green algae was tested according to OECD 201 compliant with GLP. The test organism was Pseudokirchneriella subcapitata. Due to the nature of Step 2 catalyst (UVCB), according to OECD 23 water accommodated fractions (WAF) were prepared and based on the results of a range finding tests (significant effects observed at WAF 10 and 100 mg/L) the final test was conducted at WAFs were prepared at 1.0, 3.2, 10, 32 and 100 mg/L nominal loading: individual loading rates were vigorously agitated on a magnetic stirrer for 6 days. Based on preliminary results, the period of 6 days of stirring was used to ensure that

a) Concentration of the relevant transition metal stabilizes at the pH 8.0 ± 0.3 used for ecotoxicity testing to prevent any precipitation during the test;

b) Saturation of the aquatic phase with organic hydrolysis products stemming from aromatic phosphites is obtained.

The presence of any non-dissolved test item fraction could be excluded before testing by measuring the Tyndall effect.

Analytical monitoring was performed for the relevant transition metal (ICP-MS) as well as the organic fraction (UPLC-UV at 210 nm). At WAF 1 mg/L and 100 mg/L transition metal concentration was stable over the 72 hours of the test. Step 2 catalyst concentration equivalent of 0.37 and 23.1 mg/L, respectively, was found in freshly prepared WAF (after 6 days of stirring and one day settling period). For WAF 1 mg/L, 92% of this initial concentration was recovered after 24 and 72 hours (0.34 mg/L). For WAF 100 mg/L, 100% and 103% of the initial concentration was recovered after 24 and 72 hours (23.2 / 23.7 mg/L). This is equivalent to transition metal concentrations of ca. 3 µg/L for WAF 1 mg/L and ca. 190 µg/L for WAF 100 mg/L.

With regard to the organics fraction monitored via UPLC-UV at 210 nm, initial Step 2 catalyst concentration equivalents of 1.86 mg/L and 221 mg/L were measured for WAF 1 and WAF 100 mg/L, respectively. Over the time course of the study (72 h) there was a relevant decline to 2.7 % of initial for WAF 1 mg/L and 0.2% of initial for WAF 100 mg/L. The reason for this decline is not clear. In the corresponding test for acute toxicity on Daphnia and fish an increase of absorption at 210 nm was observed. The decrease over time observed here could be either due to adsorption to algal biomass and/or further abiotic transformations (hydrolysis / oxidation) to molecular species with different chromatographic elution time / different absorption maxima during the test. The results confirm in any case that WAF preparation succeeded in a relevant organic fraction present at least at the beginning of the test.

The following results were obtained in the final test (based on growth rate inhibition):

NOELR (Pseudokirchneriella subcapitata; 72 h; static; growth rate) = 3.2 mg/L;

EL10 (Pseudokirchneriella subcapitata; 72 h; static; growth rate) = 5.9 mg/L (95% CI: 5.3-6.6)

EL50 (Pseudokirchneriella subcapitata; 72 h; static; growth rate) = 12.4 mg/L (95% CI: 11.8-13.2)