Reaction mass of dioctyl hydrogen phosphate and octyl dihydrogen phosphate

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:

2017-12-11 - 218-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted March 2006 and updated Annex 5 July 2011

GLP compliance:

yes (incl. QA statement)

Remarks:

March 2015

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.95, 7.81, 31.3, 125 and 500 mg test item/L
- Sampling method: Control and test solution were sampled in dupblicate at the start of exposure and at 72h.
- Sample storage conditions before analysis: All samples were stored in amber glass bottles in the dark at a temperature of ≤ -18°C after sampling and before shipment.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The following approach was used taking into account aspects of ISO 14442 (2006), Water quality - Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, metals and waste water, and OECD 2000, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Series on Testing and Assessment Number 23. ENV/JM/MONO(2000)6.

Stock solutions were prepared at nominal loading rates of 1.95, 7.81, 31.3, 125 and 500 mg test item/L by weighing the test item directly to the test medium for each treatment, see below. These individual stock solutions were stirred for 46 h 35 min at ambient temperature in the dark. Thereafter these stock solutions were filtered, using a glass fibre filter with an average retention capacity of 0.40 µm (MN GF-5). The initial 0.1 L were discarded. In the remaining filtrates of loading rates 1.95 to 125 mg/L no undissolved/particulate matter were visible. The 500 mg/L loading rates were filtered again because of visible particles which were subsequently not visible.

The measured pH value of the 500 mg/L loading rate filtrate (individual filtrate at a loading rate of 500 mg test item/L) was 3.3. First 500 mL were transferred to a separate volumetric flask for later exposure. Second, the remaining filtrate was adjusted to a pH of 6.2 using NaOH. The nominal loading rates of 1.95, 7.81, 31.3, and 125 mg test item/L filtrates were within the required pH range of 6–9.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: SAG 61.81
- Source: Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.8 - 22.7 °C

pH:

6.2-9.5 for nominal concentrations 1.95 to 125 mg/L
3.3 for nominal concentration 500 mg/L without pH adjustment and 5.6-6.2for nominal concentration 500 mg/L with pH adjustment

Nominal and measured concentrations:

control, 1.95, 7.81, 31.3, 125 and 500 mg test item/L (loading rates), 500 mg test item/L (loading rate) with pH adjustment
0, 1.0, 5.41, 16.9, 52.2, 234 mg/L (geometric mean measured concentration over 72h)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass Erlenmeyer flasks (300 mL) covered by air-permeable lids
- Material, size, headspace, fill volume: 100±5 mL vill volume
- Initial cells density: 0.5 × 10^4
- Control end cells density: 150.52 × 10^4
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes for nominal concentration 500 mg/L
- Photoperiod: 24h light / 0h dark
- Light intensity and quality:Mean 86.8 µE m–2s–1

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

74.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

45.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

36.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

10.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities (for algal test): None observed
In none of the concentration levels up to 125 mg test item/L (loading rates) were deformed and/or damaged algal cells observed during microscopic inspection. At the concentration level 500 mg test item/L (loading rates) no algae were observed. At the highest concentration level with pH adjustment (500 mg test item/L (loading rates)) only a few enlarged cells were observed.

The pH adjustment at the highest test concentration (loading rate of 500 mg test item/L) showed slightly lower effects than the comparable not pH adjusted concentration level. Nevertheless, the effects were meaningful compared to the control levels, thus a clear effect based on the test item properties could be stated at a concentration level at a loading rate of 500 mg test item/L (pH-adjusted).

Validity criteria
The mean biomass increase in the control cultures was at least of 16 within the 72h test period (factor was 301), the mean coefficient of variation for section-by-section specific growht rates in the control cultures was less than 35% (13.5%) and the coefficient of variation of average specific growth rates during test period in replicate control cultures was less than 7% (1%)
The study is therefore considered to be valid.

Results with reference substance (positive control):

Growth rate ErC50 (0–72 h): 0.952 mg/L
This value is within the range of the international ring test (ISO (2004). Therefore the results of this reference test are acceptable and the test conditions are reliable.

Table 1 Summary of biological results based on measured test item concentrationsof the main constituents (sum of mono- & diester) in mg main constituents/L

	Endpoints [mgmain constituents/L]^a)
Parameter	EC10	EC20	EC50	NOEC	LOEC
Yield (95% cI)	10.1 (7.80–13.2)	15.7 (12.2–20.2)	36.1 (26.4–49.0)	5.41	16.9
Growth Rate (95% cI)	45.1 (34.0–48.4)	54.4 (51.8–60.1)	74.8 (64.7–143.3)	5.41	16.9

cl: confidence limit

^a)main constituents (sum of mono- & diester)

Table 2: Summary of biological results based on nominal concentrationsin mg test item/L (loading rates).

	Endpoints[mg test item/L (loading rates)]^a)
Parameter	EC10	EC20	EC50	NOEC	LOEC
Yield (95% cI)	19.0 (17.0–20.9)	31.1 (28.7–33.5)	79.7 (75.8–83.8)	7.81	31.3
Growth Rate (95% cI)	22.9 (21.0–24.9)	51.6 (48.3–55.0)	176 (166–186)	7.81	31.3

cl: confidence limit;

^a)Individual filtrate (WAF) at different loading rates

Table 3: Summary of measured concentrations at the beginning and at the end of the test.

Conc. Level	Test period	Nominal concentration	Measured concentration	Measured concentration	Measured concentration	Recovery of nominal concentration	% of measured initial (time 0 hours) concentration	Geometric mean measured concentration over 72 h
	[d]	[mg test item/L]^a)	[mg dioctyl hydrogen phosphate/L]	[mg octyl dihydrogen phosphate/L]	[mg main constituents/L]^b)	[%*]	[%]	[mg main constituents/L]^b)
Control	0	0	<LOD	<LOD	n.d.	–	–	–
C1	0	1.95	0.72	0.57	1.29	72	–	–
C2	0	7.81	3.11	2.80	5.91	82	–	–
C3	0	31.3	9.36	10.5	19.9	69	–	–
C4	0	125	12.4	46.8	59.2	51	–	–
C5	0	500	12.5	224	237	51	–	–
Control	3	0	<LOD	<LOD	n.d.	–	–	n.d.
C1	3	1.95	0.51	0.36	0.87	48	67	1.06
C2	3	7.81	2.75	2.20	4.95	69	84	5.41
C3	3	31.3	6.47	7.84	14.3	49	72	16.9
C4	3	125	8.38	37.7	46.1	40	78	52.2
C5	3	500	11.1	220	231	50	97	234

LOQ = Limit of Quantification (1.0 mg/L); LOD = Limit of Detection(0.03 mg/L); n.d.: not determined

^a)Individual filtrate (WAF) at different loading rates

^b)Main constituents (sum of mono- & diester)

* The recoveries of nominal test item concentrations were calculated using following equation: measured concentration expressed as mg main constituents/L divided by nominal concentration expressed as mg test item/L divided by a correction factor (content of main constituents of the test item); e.g. C1: 1.29/1.95*100/(0.4332+0.4903)

Validity criteria fulfilled:

yes

Conclusions:

Expressed in terms of measured concentrations, the EC50 values for freshwater algae ranged from 36.1 to 74.8 mg/L and EC10 values ranged from 10.1 to 45.1 mg/L.

Executive summary:

The acute toxicity of the Reaction mass of dioctyl hydrogen phosphate and octyl dihydrogen phosphate to algae was tested according to OECD guideline 201 and GLP conditions.

A clear concentration-response relationship was observed for both biological parameters growth rate and yield during the exposure period.

The EbC50 of Pseudokirchneriella subcapitata was determined to be 79.7 mg test item/L (loading rates), equivalent to 36.1 mg main constituents/L (sum of mono- & diester) based on measured concentrations. The NOEC for biomass based on nominal concentration was 7.81 mg test item/L (loading rate), equivalent to 5.41 mg main constituents/L (sum of mono- & diester) based on measured concentrations.

The ErC50 of Pseudokirchneriella subcapitata was determined to be 176 mg test item/L (loading rate), equivalent to 74.8 mg main constituents/L (sum of mono- & diester) based on measured concentrations. The NOEC for growth rate based on nominal concentration was 7.81 mg test item/L (loading rate), equivalent to 5.41 mg main constituents/L (sum of mono- & diester) based on measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

74.8 mg/L

EC10 or NOEC for freshwater algae:

45.1 mg/L