Myrac Aldehyde

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:

04 Aug 2017 - 21 Sep 2017

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)

Version / remarks:

2006, corrected 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, Water Accommodated Fractions prepared at 1.0, 2.2, 4.6, 10 and 22 mg/L at t=0 h, t=24 h, t=72 h.
- Sampling volume: 4.0 mL. At the end of the exposure period, the replicates with algae were not pooled. Instead, samples were collected from one of the replicates for each test concentration.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Loading rates individually prepared at 1.0, 2.2, 4.6, 10 and 22 mg/L. A 3-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for a period of one hour. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. The entire preparation of the WAFs occurred in completely closed vessels with very limited headspace.
- Controls: Test medium without test item or other additives.
- All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata strain NIVA CHL 1
- Source: In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were 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.
- Culture medium different than test medium: yes, M1 medium.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

ACCLIMATION
- Acclimation period: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22-23 °C

pH:

At start: 7.2 - 7.3
At end: 7.6 - 8.0

Nominal and measured concentrations:

Nominal: WAFs at 1.0, 2.2, 4.6, 10, 22 mg/L
Time Weighted Average concentrations: 0.16, 0.32, 0.25, 0.80, 0.88 mg/L
For measured concentrations at t=0h, t=24h and t=72h, please refer to table 1 in section 'Any other information on results incl. tables'.
For calculation method of TWA concentrations, please refer to section 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: 40 mL all-glass vessels, airtight closed with limited headspace.
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 454 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 1 or 2 replicates of each test concentration without algae, 1 extra replicate of each test group with algae for sampling after 24 hours.
- Closed screw cap septum vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

GROWTH MEDIUM
- Standard medium used: no, Adjusted M2 medium
- Detailed composition if non-standard medium was used: Standard M2 according to the OECD 201 Guideline was adjusted with 300 mg/L NaHCO3 and 6 mmol/L HEPES buffer. pH was 7.1 ± 0.3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis)
- Culture medium different from test medium: yes, M1 medium
- Intervals of water quality measurement: pH: beginning and end of test; temperature: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuously
- Light intensity and quality: TLD-lamps with a light intensity within the range of 85 to 87 µE.m^-2.s^-1.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank.

TEST CONCENTRATIONS
- Range finding study test concentrations: Control, WAF prepared at loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes; Inhibition of growth rate:
MYRAC ALD BHT; WAFs (mg/L) Mean Std. Dev. n %Inhibition
Control 1.862 0.0028 3
1.0 1.836 0.0159 3 1.4
10 0.519 0.0898 3 72.1
100 0.331 0.0193 3 82.2

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate (performed Sep 2017)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.88 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.56 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval 0.46-0.68

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a TWA concentration of 0.88 mg/L when compared to the control.
- The analytical results of both the range-finding test and the final test suggested that the solubility of the test item in test medium is lower than the solubility in water, as given by the Sponsor (i.e., 24 mg/L). In fact, concentrations measured at the two highest test concentrations at the start of the final test were comparable, with the concentration measured in the WAF prepared at a loading rate of 10 mg/L being slightly higher than the concentration measured in the WAF prepared at a loading rate of 22 mg/L. Moreover, the concentration measured in the WAF prepared at a loading rate of 100 mg/L in the range-finding test appeared to be oversaturated, since it was observed to be slightly hazy immediately after siphoning. The consequence of the oversaturation was less effects in the final test (i.e., <50% effect for growth rate inhibition) when compared with the range-finding test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- The EC50 for growth rate inhibition (72h-ERC50) was 1.1 mg/L with a 95% confidence interval ranging from 1.1 to 1.1 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller. ECx values for were calculated with non-linear CDF (cumulative distribution fiction) regression model. The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1: Measured Concentrations Versus Nominal Concentrations

MYRAC ALD BHT WAFs (mg/L)	Measured concentration (mg/L)			TWA conc. (mg/L)
	t=0h	t=24h	t=72 h
1.0	0.505	0.255	0.016^b	0.16
2.2	1.25	0.486	0.016^b	0.32
4.6	1.01	0.487	0.00095^c	0.25
4.6^a	1.13	0.626	0.0860	0.43
10	2.88	1.44	0.024^b	0.80
22	2.11	1.31	0.19^b	0.88

a: without algae

b: estimated value, calculated by extrapolation from the calibration curve.

c: concentration was below the Limit of detection (LOD), i.e. 0.0019 mg/L. Value was calculated as LOD/2.

Table 2: Growth Rate And Percentage Inhibition For The Total Test Period

MYRAC ALD BHT TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	2.039	0.0123	6
0.16	2.016	0.0019	3	1.1^#
0.32	1.963	0.0549	3	3.8^#
0.25	1.951	0.0200	3	4.4^#
0.80	1.750	0.0733	3	14.2*
0.88	1.653	0.0328	3	18.9*

* - effect was statistically significant; # - effect was statistically significant but biologically not relevant (<10%)

Table 3: Effect Parameters

	Parameter (mg/L)	NOEC*	NOEC**	EC10	EC50
Growth rate	Value	<0.16	0.25	0.56	>0.88
	lower 95%-cl			0.46
	upper 95%-cl			0.68
Yield	Value	<0.16	0.16	0.18	0.61
	lower 95%-cl			0.12	0.39
	upper 95%-cl			0.27	1.0

* - based on statistical significance; ** - based on biological relevance

Validity criteria fulfilled:

yes

Remarks:

For details on validity criteria, please refer to section 'Overall remarks'.

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, 3-CYCLOHEXENE-1-CARBOXALDEHYDE MULTICONSTITUENT reduced growth rate of this fresh water algae species significantly at a TWA concentration of 0.80 mg/L and higher, based on biological relevance.
The EC10 for growth rate inhibition (72h-ERC10) was 0.56 mg/L, with a 95% confidence interval ranging from 0.46 to 0.68 mg/L, while the EC50 for growth rate inhibition (72h-ERC50) was > 0.88 mg/L, being considered the maximum soluble TWA concentration of the test item in test medium.

Executive summary:

The toxicity to freshwater green aquatic algae was examined in a study according to OECD TG 201 and in compliance with GLP criteria. In this study, cultures of algae (P. subcapitata) were exposed to WAFs individually prepared at loading rates of 0 (control), 1.0, 2.2, 4.6, 10 and 22 mg/L for 72 hours. Nominal target concentrations were analytically verified at the start of the test, and after 24 and 72 hours exposure. Time Weighted Average (TWA) concentrations were determined to be 0.16, 0.32, 0.25, 0.80, and 0.88 mg/L and the effect concentrations expressed as such. Cell concentrations were determined regularly throughout the test and inhibition of growth rate calculated. The test is valid.

A concentration-dependent and statistically significant inhibition of growth rate was observed. However, only the effects observed at the two highest test concentrations were considered to be biologically significant (i.e. ≥10% difference). Microscopic observations of algal cells at the highest tested concentration revealed a normal and healthy appearance. Based on these findings, the 72-h ErC50 and 72-h ErC10 values were determined to be >0.88 mg/L, and 0.56 mg/L, respectively.

Description of key information

The toxicity to freshwater green aquatic algae was examined in a study according to OECD TG 201 and in compliance with GLP criteria. In this study, cultures of algae (P. subcapitata) were exposed to WAFs individually prepared at loading rates of 0 (control), 1.0, 2.2, 4.6, 10 and 22 mg/L for 72 hours. Nominal target concentrations were analytically verified at the start of the test, and after 24 and 72 hours exposure. Time Weighted Average (TWA) concentrations were determined to be 0.16, 0.32, 0.25, 0.80, and 0.88 mg/L and the effect concentrations expressed as such. Cell concentrations were determined regularly throughout the test and inhibition of growth rate calculated. The test is valid.

A concentration-dependent and statistically significant inhibition of growth rate was observed. However, only the effects observed at the two highest test concentrations were considered to be biologically significant (i.e. ≥10% difference). Microscopic observations of algal cells at the highest tested concentration revealed a normal and healthy appearance. Based on these findings, the 72-h ErC50 and 72-h ErC10 values were determined to be >0.88 mg/L, and 0.56 mg/L, respectively.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.56 mg/L