2,4,7,9-Tetramethyldec-5-yne-4,7-diol, ethoxylated

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

Remarks:

study is ongoing, start of the main study (experimental part) November 2022

Adequacy of study:

key study

Study period:

Oct-Dec 2022

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:

23. March 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number: NP20701763
- Origin: Evonik Operations GmbH
- Purity: 100 %

Expiration date:.17 July 2024

Analytical monitoring:

yes

Details on sampling:

Verification of test concentrations
Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20°C for further analysis if necessary.
The stability of the test item under storage conditions was verified by a pre-experiment.

Determination of Algal Biomass
A small volume (100 µL per sample) of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced.

The algal biomass in the samples was determined by fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK). The measurements were performed at least in duplicate at an excitation of 440 nm and emission of 680 nm.

At the end of the test, a sample was taken from the control and from the nominal concentration of 100 mg/L to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were microscopically inspected.

Analysis of the Test Item Concentrations
For the determination of the actual test item concentrations, duplicate samples were taken from each treatment at the start of the test.

For sampling at the end of the test, the test medium of the treatment replicates was pooled.

All samples were stored frozen (at -20 ± 5 °C) immediately after sampling. Based on analytical pre-experiments for investigation of the storage stability, the test item was found to be stable in the test water under these storage conditions.

The concentrations of CAD-2021-941 were analytically measured in one of the duplicate test medium samples from the nominal test concentrations of 0.32 mg/L to 100 mg/L per sampling time. From the control samples, one of the duplicate samples was analyzed per sampling time.

Vehicle:

yes

Remarks:

reconstituted water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Preparation for Range-finding test
The test concentrations were 1.0, 10 and 100 mg/L. For preparation of the highest test concentration 102.4 µL test item were dissolved in 1000 mL test water using intensive stirring for 15 minutes. This volume is equivalent to a concentration of 100 mg/L, considering the density of the test item of 0.98 g/cm3. The two lower test concentrations were prepared by serial dilution steps.

The analytical results demonstrated the stability of the test item during the test for all treatments with 101 % of nominal concentrations measured at the start of the test and 95 to 102 % after 72 hours test duration.

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preparation for Definitive test
Based on the results of the range-finding test, nominal test item concentrations of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (spacing factor 3.2) were tested in the main test. Additionally, a control (test water without test item) was be run in parallel.

- Method: Reconstituted test water (AAP medium) prepared according to the test guidelines was used for cultivation of the algae and testing. Analytical grade salts were dissolved in sterile purified water to obtain the following concentrations:

Ingredients Concentration
Macro-nutrients
NaHCO3 15.0 mg/L
K2HPO4 1.044 mg/L
MgSO4 × 7 H2O 14.6 mg/L
MgCl2 × 6 H2O 12.16 mg/L
CaCl2 × 2 H2O 4.41 mg/L
NaNO3 25.5 mg/L
Trace elements
H3BO3 186.0 µg/L
MnCl2 × 4 H2O 415.0 µg/L
ZnCl2 3.27 µg/L
CoCl2 × 6 H2O 1.43 µg/L
CuCl2 × 2 H2O 0.012 µg/L
Na2MoO4 × 2 H2O 7.26 µg/L
FeCl3 × 6 H2O 160.0 µg/L
Na2EDTA × 2 H2O 300.0 µg/L

The test medium of the highest nominal concentration of 100 mg/L was prepared by mixing 102.04 µL of the test item into 1000 mL of test water using intense stirring for 15 minutes at room temperature. This volume is equivalent to a concentration of 100 mg/L, considering the density of the test item of 0.98 g/cm3. No auxiliary solvent or emulsifier was used.

This highest test concentration was used in a series of dilutions with test water to obtain the test media with the lower test concentrations. All solutions were clear with no evidence of undissolved test item. The test media were prepared just before the start of the test.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Microalga, Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum, occasionally also listed as Raphidocelis subcapitata)
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae are cultivated at IES Ltd Laboratories under standardized conditions according to the test guidelines.
- Age of inoculum (at test initiation): An inoculum culture was set up four days before the start of the exposure.
- Method of cultivation: The algae were cultivated under the test conditions.

ACCLIMATION
- Acclimation period: The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.
- Culturing media and conditions (same as test or not): AAP Medium. The algae were cultivated under the test conditions.
- Any deformed or abnormal cells observed: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 100 mg/L (mean measured 88 mg/L) and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

Test type:

static

Water media type:

other: Reconstituted test water (AAP medium) prepared according to the test guidelines

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

0.15 mmol/L (= 15 mg/L as CaCO3)

Test temperature:

21-24°C

pH:

7.3

Dissolved oxygen:

not determined

Salinity:

Please refer to details on test solution

Conductivity:

not determined

Nominal and measured concentrations:

Based on the results of the range-finding test, nominal test item concentrations of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (spacing factor 3.2) will be tested in the main test. Additionally, a control (test water without test item) will be run in parallel.
measured concentrations of the dose range finder study are between 95 and 102% of the nominal concentrations after 0 and 72 hours.
Details are given in the field 'any other information on materials and methods incl. tables.

The measured concentrations of the test item CAD-2021-941 in the test media are shown in the table below and in Table 6, Appendix 1. The measured concentrations of CAD-2021-941 in the test media of all test concentrations were between 84 and 87 % of the nominal values at the start of the test. Thus, the correct dosage of the test item could be verified. The test item concentrations in the test media aged samples were ranged from 78 % to 93 % at day 3 (72 hours). Since the test item concentrations slightly decreased over the test period for some of the test concentrations, the mean measured concentrations were calculated as the geometric mean of the concentrations measured at both sampling dates (0 and 72 hours).

Nominal Analytically Measured Mean Measured
Test Item Concentration Concentration
Concen of the Test Item [mg/L] (Geometric Mean) [mg/L]
tration
0 Hours 72 Hours
0.32 0.269 0.250 0.26
1.0 0.846 0.813 0.83
3.2 2.80 2.80 2.8
10 8.59 8.65 8.6
32 27.8 29.7 29
100 85.5 90.0 88

The biological results are based on the mean measured test item concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 75 mL Erlenmeyer flasks
- Type (delete if not applicable): Each test flask was loosely covered with a glass lid, thereby adequate CO2 could be transferred from the surrounding air to the test solution.
- Material, size, headspace, fill volume: The volume of test solution in each test flask was 30 mL per replicate.
- Aeration: The test flasks were incubated in an orbital shaker (Multitron-Pro, Infors HT, Bottmingen/Switzerland)
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: The test was started using a nominal algal cell density of 5000 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates):


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (AAP Medium) prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water. Refer to the attached tables for details.
- Intervals of water quality measurement: The temperature in the incubator was monitored and recorded continuously. The appearance of the test media was also visually controlled and recorded daily during the exposure period.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH was measured and recorded in each treatment at the start and end of the test.
- Photoperiod: The test flasks were continuously illuminated by LED light installed above the test flasks.
- Light intensity and quality: The mean light intensity at the level of the test solutions was approximately 65 µE.s-1.m-2 (range: 63 to 66 µE s-1 m-2, measured at nine places in the experimental area). The light intensity over the incubation area was within a ±15 %-deviation from the average light intensity as recommended by the guideline.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (Cell Counter CASY TT, OLS, Bremen/Germany)

TEST CONCENTRATIONS
- Range finding study
For determination of the appropriate test concentrations for the main test, a range finding test (without GLP) was performed. For this, three replicates per treatment were tested in parallel with a control.

Test procedure and conditions were comparable to those applied in the main test with the following exceptions: For the control, three replicates were prepared.

The test concentrations were 1.0, 10 and 100 mg/L. For preparation of the highest test concentration 102.4 µL test item were dissolved in 1000 mL test water using intensive stirring for 15 minutes. This volume is equivalent to a concentration of 100 mg/L, considering the density of the test item of 0.98 g/cm3. The two lower test concentrations were prepared by serial dilution steps.

The analytical results demonstrated the stability of the test item during the test for all treatments with 101 % of nominal concentrations measured at the start of the test and 95 to 102 % after 72 hours test duration.

For a better understanding of the behavior and fate of the test item under the test conditions, additional test vessels with test medium at a concentration of 10 mg/L were incubated for 72 hours under the following conditions:
- Test medium without algae under light conditions
- Test medium with algae in a closed vessel

The analytical results for these additional replicates showed no significant difference compared to the test conditions.

- Test concentrations: 0.32, 1.0, 3.2, 10, 32, and 100 mg/L
- Results used to determine the conditions for the definitive study:

CULTURING APPARATUS
-Details on culturing apparatus used: Multitron-Pro, Infors HT, Bottmingen/Switzerland

Reference substance (positive control):

yes

Remarks:

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 4.4 mg/L

95% CI:

>= 2.9 - <= 6.8

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 7.5 mg/L

95% CI:

>= 5 - <= 11

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 20 mg/L

95% CI:

>= 12 - <= 33

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 18 mg/L

95% CI:

>= 16 - <= 20

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 30 mg/L

95% CI:

>= 26 - <= 34

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 78 mg/L

95% CI:

>= 65 - <= 91

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.83 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 2.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): In the control, the biomass increased by a factor of 125 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled.
- Observation of abnormalities (for algal test): The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 100 mg/L (mean measured 88 mg/L) and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

Results with reference substance (positive control):

The 72-hour EC50 for growth rate in the reference test IES Study Number 20220372 was 0.76 mg/L (October 2022) and showed that the sensitivity of the test system was slightly higher than the range recommended by the guideline (72-hour EC50 for the growth rate 0.92 1.5 mg/L).

Reported statistics and error estimates:

Inhibition of algal growth was determined from the following growth parameters:

a) the specific growth rate (µ)
b) the yield (Y)

using the following equations:

a) Specific growth rate (µ):

µi-j=(lnXj-lnXi)/(tj-ti)

where: µi–j: average specific growth rate from time i to j
Xi: biomass at time i
Xj: biomass at time j


The average growth rate over the test duration and the section-by-section growth rates (daily growth rates between the sampling times) were calculated.

Inhibition of growth rate (Ir):

Ir=(µc-µr)/µc*100%

where: Ir: percent inhibition in average specific growth rate
µC: mean value for average specific growth rate in the control group
µT: average specific growth rate for the treatment replicate

b) Yield (Y):

Y=Xj-X0

where: Y: yield
X0: biomass (nominal value) at the start of the test
Xj: biomass at time j (at the end of the test)


Inhibition of yield (Iy):

Iy=(Yc-Yr)/Yc*100%

where: Iy: percent inhibition of yield
YC: mean value for yield in the control group
YT: value for yield for the treatment replicate

Growth rate and yield were calculated for each test flask. The mean values for growth rate µ and yield Y were calculated for each treatment.

The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95 % confidence intervals were calculated by three parameter normal cumulative distribution function.

For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test.

Statistical analysis was performed using ToxRat Professional®

The results of the range finding experiment were as follows:

 

Nominal Test Item Concentration mg/L	Inhibition of Yield  after 72 Hours	Inhibition of Growth rate  after 72 Hours
	[%]	[%]
Control	0	0
1.0	7.8	1.7
10	26	6.3
100	95	66

Validity criteria fulfilled:

yes

Conclusions:

A clear concentration-response relationship was observed for both biological endpoints growth rate and yield after the exposure period of 72 hours.
The 72h-EC50 was ca.78 mg/L.

Executive summary:

The impact of the test item CAD-2021-941 on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72‑hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No 2016/266, C.3.

 

The nominal test item concentrations tested were 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel.

 

The measured concentrations of CAD-2021-941 in the test media of all test concentrations were between 84 and 87 % of the nominal values at the start of the test. Thus, the correct dosage of the test item could be verified. The test item concentrations in the test media aged samples were ranged from 78 % to 93 % at day 3 (72 hours). Since the test item concentrations slightly decreased over the test period for some of the test concentrations, the mean measured concentrations were calculated as the geometric mean of the concentrations measured at both sampling dates (0 and 72 hours).

A clear concentration-response relationship was observed for both biological endpoints growth rate and yield after the exposure period of 72 hours.

Description of key information

72 h ErC10 = 18 mg/L (95% c.i. 16 to 20 mg/L)

72 h EC50 = 78 mg/L (95% c.i. 65 to 91 mg/L)

 

Key value for chemical safety assessment

EC50 for freshwater algae:

78 mg/L

EC10 or NOEC for freshwater algae:

18 mg/L