Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate

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:

From November 2017 to April 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Appearance: Colourless to brown liquid
- Test item name: Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate
- Synonym: lnnroad Protect
- Expiration date of the lot/batch: 01 June 2019
- Purity: 98.2% (Di-ester content)


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep container tightly closed in a dry and wellventilated place
- Solubility in water: Highly soluble (estimation: 20-50 g/L)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: in the control and all test solutions.
- Sampling frequency: At start (0 hours) and at the end of exposure (72 hours). Control and test solutions were sampled in duplicate at the start of exposure without algae and at the end of exposure with algae and without algal (additional stabilty control). The duplicate samples were kept separately as a reserve.
- Sample storage conditions before analysis: The samples were stabilised by adding acetonitrile directly after sampling (acetonitrile volume: 20% of the water sample volume, 10 mL of sample were mixed with 2 mL of acetonitrile). After sampling and before shipment, all samples were stored in glass bottles in the dark at a temperature of 5 -18°C

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution was prepared by dissolving the test item in test medium A stock solution at a loading rate of 0.1008 g test item per 1000 ml was prepared by directly adding the test item to the test medium. This stock solution was stirred (700 rpm) using a magnetic stirrer for 15 min at ambient temperature in the dark. Thereafter this stock solution was left to settle for 40 minutes. The stock solution was visually examined for undissolved/particulate matter in the free water column. No undissolved/particulate matter was
observed and therefore stock solution was used for preparation of test solutions.

After temperature adaptation of the test solutions, 3.488, 1.744 or 1.081 mL of the algal preculture (Raphidocelis subcapitata) were added to each of the volumetric flasks containing
1000, 500 or 310 mL of the test solutions, respectively to achieve an algal cell concentration of approximately 0.3 x 10x4 cells/mL. These manipulations were performed under a laminar flow box. The test solutions were homogenised by manual shaking 15 times overhead, before adding 100±5 mL of the test media to each test vessel to ensure a homogeneous distribution of the algal cells.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata species name: Raphidocelis subcapitata
- Strain: (SAG 61.81), currently valid
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany

PRE-CULTURE CONDITIONS:
- Culture medium: OECD medium (modified)
- Volume of liquid stock cultureper pre-culture vessel: 100±5 mL
- Pre-culture vessels: 300 mL Erlenmeyer flasks
- Number of replicates: 2
- Light: Permanent illumination (24/0 h light/dark); Econlux LED Sunstrip "daylight"
- Light intensity: 60-120 pE m 2s-1; (measured: 70.5-73.6 pE m 2s-1)
- Shaker: 100±5 oscillations/min
- Temperature in the test room: 21-24 °C; (measured: 21.8-22.2 °C)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

none

Test temperature:

21.8 - 22.0°C

pH:

From 7.5 to 8.0 in control
From 6.6 to 7.7 or 7.5 to 8.1 in the test solutions

Nominal and measured concentrations:

Nominal concentrations: control, 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L based on the results of a preliminary non-GLP range finding test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass Erlenmeyer flasks (300 mL) covered by air-permeable lids
- Type: closed
- Age of the pre-culture: 3 days
- Agitation: 100±5 oscillations/min; the test vessels were placed randomly on a horizontal shaker, so that each test vessel was rotated around its own axis
- Initial cells density: 0.3 x 10e4 cells
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Number of replicates for stability check (highest concentration level without algae): 1


TEST MEDIUM:
- Test medium: OECD medium (modified)
- pH of test medium: 8.3

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24 h light/0 h dark
- Light intensity and quality:72.8-84.2 µE/m2/s;mean 77.1 µE/m2/s


EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: At start of the test, the cell numbers were determined microscopically using a counting chamber (Thoma chamber). After 24, 48 and 72 hours, the cell numbers were determined by measuring the fluorescence intensity in 4 samples of 200 pL of test solution per replicate using a fluorometer (Multiple Reader Tecan ULTRA).
- pH: pH was measured and recorded for each test concentration and the control.at the start of the test After temperature adaptation of the test solutions and t the end of the test, the pH was measured and recorded in the pooled replicates per test concentration and control.
- Temparature : Temperature was recorded once per hour throughout the test in a separate test vessel placed on the shaker containing approximately 100±5 mL deionised water.


TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: control, 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L based on the results of a preliminary non-GLP range finding test.

Reference substance (positive control):

yes

Remarks:

A reference test using potassium dichromate (K2Cr207) as reference item were performed in October 2017

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

46.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

ANALYTICAL RESULT
At time 0 hours, the measured concentrations ranged between 96-108% of the nominal concentrations. After 72 hours exposure, the test concentrations decreased to levels of For each concentration level the geometric mean concentration was calculated using the test item concentrations measured at the start (0 hours) and the end of the exposure period (72 hours). Concentrations determined to be The following geometric mean concentrations were determined: 1.74, 2.48, 3.28, 14.0, and 35.2 mg test item/L.

BIOLOGICAL RESULTS:
A clear concentration-response relationship was observed for the biological parameter yield during the exposure period, and a weak concentration-response relationship was observed for the biological parameter growth rate.
- Inhibition of Growth Rate: The EC50 for growth rate of Raphidocelis subcapitata was stated to be >100 mg test item/L, equivalent to > 35.2 mg Reaction mass of diethyl adipate and diethyl glutarate and diethyl
succinate/L based on measured concentrations. The NOEC based on nominal concentration appeared to be 25.0 mg test item/L, equivalent to 3.28 mg Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate/L based on measured concentrations. (see table 1)

- Inhibition of Yield: The EC50 for yield of Raphidocelis subcapitata was stated to be >100 mg test item/L, equivalent to >35.2 mg Reaction mass of diethyl adipate and diethyl glutarate and diethyl
succinate/L based on measured concentrations. The NOEC based on nominal concentration appeared to be 25.0 mg test item/L, equivalent to 3.28 mg Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate/L based on measured concentrations. (see table 2)

Observations
In all of the concentration levels (control and test solutions), no deformed and/or damaged algal cells were observed during microscopic inspection.

Reported statistics and error estimates:

The data were evaluated by the Shapiro-VVilk's Test for normal distribution, and by Levene's Test for homogeneity of variances. The Williams Multiple Sequential t-test was applied to find out whether there were significant differences between the growth of algae in the controls and the algae exposed to the test item concentration.
Non-linear regression analyses (3-param. normal CDF analyses) was used to determine of the concentration-response function.
The statistical software package ToxRat 3.2 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.
The statistical analysis was performed using nominal concentrations as well as mean measured concentrations.

Table 1 : Inhibition of Growth Rate: Growth Rate of Raphidocelis subcapitata in relation to concentration of the test item and %Inhibition caused by the test item, after 72 hours.

Nominal concentration [mg/L]	Mean growth rate [day-1]	Std. Dev. [day-1]	n	%Inhibition
Control	1.866	0.0205	6	-
6.25	1.853	0.0230	3	0.7
12.5	1.852	0.0066	3	0.7
25.0	1.873	0.0224	3	-0.4
50.0	1.812	0.0070	3	2.9*
100	1.714	0.0064	3	8.01*

 * Significant difference from control (Williams test; p ≤0.05)

Table 2 : Inhibition of Yield: Yield per mL (divided by 10000) of Raphidocelis subcapitata in relation to concentration of the test item and %Inhibition caused by the test item, after 72 hours.

Nominal concentration [mg/L]	Mean Yiel [day-1]	Std. Dev. [day-1]	n	%Inhibition
Control	80.7	5.05	6	-
6.25	77.8	5.29	3	3.6
12.5	77.4	1.54	3	4.1
25.0	82.6	5.53	3	-2.4
50.0	68.6	1.45	3	15.0*
100	51.0	0.98	3	36.7*

 * Significant difference from control (Williams test; p ≤0.05)

Validity criteria fulfilled:

yes

Conclusions:

At the highest tested concentration level (100 mg/L), the percentage inhibition was <40% for the yield and was <10% for the growth rate. Therefore, the EyC50 (yield), the ErC50 and the ErC10 (growth rate) were stated to be >100 mg/L, equivalent to >35.2 mg Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate/L based on measured concentrations.
The NOEC for the growth rate and yield based on nominal concentration appeared to be 25.0 mg test item/L, equivalent to 3.28 mg Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate/L based on measured concentrations.

Executive summary:

The toxic effect of the test item on the growth of the freshwater green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was investigated in a 72‑hour static test according to OECD Guideline 201 (2006; Annex 5 corrected 28 July 2011) The study was compliant with the GLP. The objective was to determine the NOEC, EC10 and EC50 for both inhibition of growth rate and inhibition of yield.

 

Based on results of a preliminary range-finding test, Raphidocelis subcapitata was exposed to the test item over a range of nominal concentrations of 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L (three replicate flasks per concentration) and a control (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24± 1 °C. Two additional test vessels with test solutions at 100 mg//l without algae were performed for stability purposes, one protected from light.

Samples of the algal populations were removed daily and Algal biomass determined for each control and test solutions.

Samples from the test solutions were analysed to determine actual levels of the test item in solution at start of exposure period (time 0 hours) and at the end of the exposure at 72 hours.

At time 0 hours the measured concentrations range between 96-108% of the nominal concentrations. After 72 hours exposure, the test concentrations decrease to levels of < LOD (Limit of Detection - 12% of the nominal concentrations. Since the measured concertation remained not stable within ±20% throughout the exposure period, the biological results are also reported based on geometric mean measured concentrations at each concentration level.

 

Stability samples without algae showed no decline of test item concentration throughout the exposure period. The determined recovery was 93% (test solutions at 100 mg test item/L) of nominal concentration. This indicates that the decrease of test item is correlated to the presence of algae cells in the test solutions.

 

At the highest tested concentration level (100 mg/L), the percentage inhibition was <40% for the yield and was <10% for the growth rate. Therefore, the EyC50 (yield), the ErC50 and the ErC10 (growth rate) were stated to be >100 mg/L, equivalent to >35.2 mg Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate/L based on measured concentrations.

The NOEC for the growth rate and yield based on nominal concentration appeared to be 25.0 mg test item/L, equivalent to 3.28 mg Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate/L based on measured concentrations.

 

The three validity criteria of the OECD guideline 201 were fulfilled, therefore this study is considered as reliable without restrictions.

Description of key information

The toxicity of the Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate to the freshwater green algae Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 201.

Under the conditions of the study, at the highest tested concentration level (100 mg/L), the percentage inhibition was <40% for the yield and was <10% for the growth rate. Therefore, the EyC50 (yield), the ErC50 and the ErC10 (growth rate) were stated to be >100 mg/L, equivalent to >35.2 mg Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate/L based on measured concentrations.

The NOEC for the growth rate and yield based on nominal concentration appeared to be 25.0 mg test item/L, equivalent to 3.28 mg Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate/L based on measured concentrations

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The toxicity of the test item to freshwater green algae Pseudokirchneriella subcapitata was investigated in one GLP-compliant study performed in accordance with standard methods, without deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.