Calcium hydrogen phosphonate

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:

08-26-16 to 12-13-16

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)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

After 24 and 48h of incubation, aliquot of 200 µL was sampled from each inoculated test flask, pipetted into a microplate. After 72 h, the samples with high cell density were diluted to ¼ (50 µL + 150 µL of filtered dilution water) and pipetted into a microplate.
Analysis were performed immediately after sampling.

Vehicle:

no

Details on test solutions:

For both range-finding test and definitive tests, tests solutions were prepared from dilutions of a saturated stock solution.
A known weight of test item (0.9997g for the preliminary range-finding test and 1.0052g for the definitive test) was poured into a volumetric flask, the volume was then made up to 1000 mL with test medium.
The solution was kept under high speed stirring (750 to 1500 rpm) at ambient temperature during a few minutes with a magnetic stir bar.
Due to the presence of undissolved particles, this solution was filtered through 0.45 µm filter before dilution.

Test organisms (species):

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

Details on test organisms:

The test organism used for the study was Pseudokirchneriella subcapitata, Strain No. CCAP 278/4, supplied by the Culture Centre of Algae and Protozoa (Ambleside, UK).

Transfers of P. subcapitata were made regularly to provide suitable subcultures. The algae were cultivated under standardized conditions as described in Annex 2 of the OECD 201 guideline.

The quality of the stock culture was checked for the absence of micro-organisms and deformed or abnormal cells under microscopic observation before use.

Four days before the start of the exposure, two pre-cultures were prepared by inoculating sufficient cells from the algal stock culture to the growth medium to obtain a low cell density of 1.104 cells/mL for pre-culturing, in order to maintain exponential growth until the start of the test. These pre-cultures were incubated under the same conditions as those used for the test cultures. Only one of the two pre-cultures was used to inoculate the test flasks for the study. The second one was only used if the first one was damaged.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Mean : 22.7 °C

pH:

7.9 - 9.1

Dissolved oxygen:

8.6 - 9.2 mg/L

Nominal and measured concentrations:

Nominal concentrations (% v/v saturated solution): 0 - 3.2 - 10.0 - 32.0 - 100

Initial measured concentrations (mg/L) : 0 - 1.4 - 4.5 - 14.4 - 45.8 - 140.6

Final measured concentrations (mg/l) : 0 - 1.4 - 4.6 - 14.4 - 45.6 - 138.8

Details on test conditions:

The incubation was conducted in a phytoculture cabinet that allows test flasks to be incubated under precise conditions: temperature was set to 23 ± 1°C.

Flasks were continuously shaken with a rotation at around 100 rpm and constantly illuminated by fluorescent tubes between 6,000 and 10,000 lux.

Test vessels were made of glass bottles (120 mL capacity) capped with cellulose bungs. They were filled with a volume of 50 mL.

At the beginning of the test, the cell density of the pre-culture was determined. The result was used to calculate the volume to be introduced into each test flask in order to obtain an initial cell concentration of 1104 cells/mL as recommended in the OECD 201 guideline.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

139.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

Solutions were found to be clear, no precipitation was observed at the end of the test.

Microscopic observation : exposed algae appeared inflated and shorter than controls.

Concentrations of the test item were maintained over the 72h test period (i.e. deviation < 20%). As a consequence, the stability of the substance was confirmed over the test period. The exposure concentrations were thus based on the initial measured concentrations.

Exponential growth of control culture at the expected rate throughout the test.

The results (growth rate) demonstrated that algal growth was inhibited from 1.4% at the lowest concentration to 53.8% at the highest tested concentration.

72h-ErC50 : 139.9 mg/L
72h-ErC10 : 5.8 mg/L

Results with reference substance (positive control):

Algal growth inhibition test on potassium dichromate.

The latest value of ErC50 obtained in August 2016 was 0.81 mg/L.

For information, according to ISO 8692 standard, 72h-ErC50 obtained from an inter-laboratory exercise on potassium dichromate was in the range 0.65 to 1.73 mg/L.

Reported statistics and error estimates:

The growth inhibition data were analysed using an Excel program. EC10 and EC50 values as well as the 95% confidence intervals were then determined using the software ToxRat® V3.1 according to the recommendations of the guideline.
As there was evidence that the concentration of the substance being tested were satisfactorily maintained within ± 20% of the measured initial concentration throughout the test, analysis of the results was based on measured initial values.

Test solution	Replicate	Algal density at 24h (Fd, ¢ *10^4/ ml )			Algal density at 48h (Fd, ¢ *10^4/ ml )			Algal density at 72h (Fd, ¢ *10^4/ ml )			Average specific growth rate J0-J3				Specific growth rate inhibition (%)
		Replicate mean	Mean	RSD	Replicate mean	Mean	RSD	Replicate mean	Mean	RSD	Replicate mean	Average	Sx	CV
Control	a	7.34	6.71	7.53	28.58	27.96	2.12	144.89	141.07	8.48	1.659	1.649	0.028	1.707	Inhibition (per replicate)	Inhibition (mean)
	b	6.05			27.72			129.73			1.622
	c	6.94			28.20			155.51			1.682
	d	6.30			26.92			133.39			1.631
	e	6.49			27.96			129.10			1.620
	f	7.13			28.37			153.81			1.679
1.00	a	6.96	6.79	4.27	31.07	29.71	5.09	134.04	131.40	1.98	1.633	1.626	0.007	0.406	1.0	1.4
	b	6.45			28.08			128.83			1.620				1.8
	c	6.94			29.99			131.33			1.626				1.4
3.20	a	7.52	6.72	10.27	30.57	26.03	15.11	129.22	97.05	28.92	1.621	1.516	0.091	6.019	1.7	8.0
	b	6.34			23.68			84.46			1.479				10.3
	c	6.31			23.83			77.49			1.450				12.1
10.00	a	5.73	5.41	7.94	18.98	18.30	14.86	38.41	40.09	32.86	1.216	1.218	0.111	9.075	26.2	26.1
	b	4.92			15.30			27.83			1.109				32.8
	c	5.57			20.61			54.02			1.330				19.3
32.00	a	4.46	4.31	3.05	12.87	11.96	6.75	19.58	18.37	6.06	0.992	0.970	0.020	2.067	39.9	41.2
	b	4.28			11.66			17.39			0.952				42.3
	c	4.21			11.35			18.14			0.966				41.4
100.00	a	3.57	3.39	4.97	7.10	6.84	6.72	10.66	9.85	7.42	0.789	0.762	0.024	3.204	52.2	53.8
	b	3.33			7.12			9.24			0.741				55.0
	c	3.25			6.31			9.64			0.755				54.2

Validity criteria fulfilled:

yes

Conclusions:

This study was designed to determine the effects of the test item on the growth of Pseudokirchneriella subcapitata in a 72-hour test according to the OECD 201 Guideline.

All validity criteria were fulfilled.

72h-ErC10 = 5.8 mg/L
72h-ErC50 = 139.9 mg/L

Executive summary:

This study was designed to determine the effects of the test item on the growth of Pseudokirchneriella subcapitata in a 72-hour test according to the OECD 201 Guideline.

For both range-finding test and definitive test, tests solutions were prepared from dilutions of a saturated stock solution.

Chemical analysis of the test item showed the stability of the substance over the 72-hour test period.

- Initial measured concentrations (mg/L) : 0 - 1.4 - 4.5 - 14.4 - 45.8 - 140.6

- Final measured concentrations (mg/l) : 0 - 1.4 - 4.6 - 14.4 - 45.6 - 138.8

Based on these results, the exposure concentrations were based on the initial measured concentrations.

Results are reported as growth rate inhibition. The concentrations that resulted in a 10 and 50% reduction in growth rate were determined as follows:

72h-ErC10 = 5.8 mg/L

72h-ErC50 = 139.9 mg/L

 

 The definitive test met the validity criteria of the test guideline detailed as follows:

 - The biomass in the control cultures increased exponentially by a factor of 141.1 within the 72-hour test period (required: at last 16-fold)

 - The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures was max 14.6% (required: < 35%)

 - The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 1.7% (required: < 7%)

Description of key information

This study was designed to determine the effects of the test item on the growth of Pseudokirchneriella subcapitata in a 72-hour test according to the OECD 201 Guideline.

For both range-finding test and definitive test, tests solutions were prepared from dilutions of a saturated stock solution.

Chemical analysis of the test item showed the stability of the substance over the 72-hour test period.

- Initial measured concentrations (mg/L) : 0 - 1.4 - 4.5 - 14.4 - 45.8 - 140.6

- Final measured concentrations (mg/l) : 0 - 1.4 - 4.6 - 14.4 - 45.6 - 138.8

Based on the chemical analysis results, the exposure concentrations were based on the initial measured concentrations.

Results are reported as growth rate inhibition. The concentrations that resulted in a 10 and 50% reduction in growth rate were determined as follows:

72h-ErC10 = 5.8 mg/L

72h-ErC50 = 139.9 mg/L

 

 The definitive test met the validity criteria of the test guideline detailed as follows:

 - The biomass in the control cultures increased exponentially by a factor of 141.1 within the 72-hour test period (required: at last 16-fold)

 - The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures was max 14.6% (required: < 35%)

 - The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 1.7% (required: < 7%)

Key value for chemical safety assessment

EC50 for freshwater algae:

139.9 mg/L

EC10 or NOEC for freshwater algae:

5.8 mg/L