Sodium benzoate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01/12/2021 to 29/06/2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Substance Name: Purox® S Grains Pure Grade Sodium Benzoate (will be referred to as Sodium Benzoate for study purposes)

CAS No.: 532-32-1
Chemical Formula: C7H6O2.Na
Molecular Weight: 144.1 g/mol
Lot Number: 2107-2
Internal Number: ESTS 229/21
Purity: 99.99%
Appearance: White Granules
Receipt Date: 27 September 2021
Expiry Date: 15 February 2024
Storage: Room temperature (15-25°C)

Analytical monitoring:

yes

Details on sampling:

Range-finding Test:
The range-finding test was conducted using nominal concentrations of 0.01, 0.1, 1.0 and 10 mg/L. A control group was also included. Only the key findings have been reported herein.
The concentrations for the range-finding test were prepared by successive 1:10 serial dilutions from a 10 mg/L stock solution (highest concentration). The 10 mg/L stock solution was prepared by adding ca 5 mg (5.01 – 5.06 mg) of test substance to 500 mL of Elendt M4 media. The 10 mg/L stock solution was then sonicated for ca 30 seconds, as required.

The control solution was prepared with Elendt M4 media only.
Duplicate test vessels were prepared for the control and each test concentration.
One juvenile Daphnia magna, less than 24 hours old, was added to each test vessel, using a wide bore pipette to avoid damaging the animals during transfer. The D. magna were fed daily with a suspension of Chlorella vulgaris (CCAP 211/12).
The range-finding test was stopped on Day 13 after all treatments had their second brood.


Definitive Test:
Based on the results of the range-finding test, the definitive test was conducted at the following nominal concentrations: 0.1, 0.32, 1, 3.2 and 10 mg/L. A control group was also included in the test.

Vehicle:

yes

Remarks:

The dilution water used in the study was Elendt M4 medium

Details on test solutions:

Preparation of Test Medium:

The 10 mg/L test solution used for the definitive test was prepared by adding ca 10 mg (10.00 - 10.03 mg) of test substance to 1000 mL of Elendt M4 medium (test media code = TM1). The solution was sonicated for ca 1 minute to aid dissolution.

TM1 was then used to prepare the other test solutions, as detailed in the table below: 

Test Media Code (mg/L) Volume of Test Media Used Final Volume (mL) Nominal Concentration

TM2 320 mL of TM1 1000 3.2
TM3 313 mL of TM2 1000 1
TM4 320 mL of TM3 1000 0.32
TM5 313 mL of TM4 1000 0.1


The control treatment was prepared with Elendt M4 medium only.

Test organisms (species):

Daphnia magna

Details on test organisms:

Juvenile Daphnia magna (less than 24 hours old) were taken from healthy laboratory cultures maintained in media of the same composition and quality as that used in the toxicity tests. These cultures were set up originally from dormant eggs (ephippia) supplied by MicroBioTests Inc., Belgium

Feeding
The Daphnia magna were fed daily with a suspension of Chlorella vulgaris (CCAP 211/12). The daily feeding rate of Chlorella vulgaris was between approximately 1.987 x 107 and 3.987 x 107 cells per vessel per day to provide each Daphnia magna with between approximately 0.1 and 0.2 mg carbon/day (cell concentration ranges were calculated using recent total organic carbon (TOC) analysis data for the Chlorella vulgaris culture (10 December 2020)).
The amount of algae added to each test vessel was based on the age of the Daphnia magna and on the amount of precipitated algae present on the base of the vessels or in suspension directly before feeding.

Observations
The adult Daphnia magna were observed for immobility, the presence or absence of eggs developing in the brood pouch (gravid or non-gravid) and mortality daily throughout the duration of the test. In addition, the number of juveniles present (alive or dead) were recorded daily, then removed from the vessels and discarded.
A Daphnia magna was considered immobile if, when the contents of the test vessel were briefly agitated, it did not swim during a 15-second period of observation.
If the Daphnia magna did not survive to Day 21, any juveniles produced by the parent, during the test, were excluded from the analysis.

Growth Measurements
At the end of the test, the carapace lengths of all surviving parental Daphnia magna were measured using a microscope fitted with a graticule slide.
The carapace measurement was taken from the top of the head to the base of the anal spine. The anal spine was not measured as it can vary in length between moults

Test type:

semi-static

Water media type:

other: Elendt M4 medium

Limit test:

yes

Total exposure duration:

21 d

Test temperature:

Continuously recorded using a digital maximum / minimum thermometer held within the test area
min: 20.2
max: 21.1

pH:

between 7.51 and 8.22

Dissolved oxygen:

between 83.4 and 102.9

Nominal and measured concentrations:

nominal concentrations of 0.1, 0.32, 1, 3.2 and 10 mg/l
Geometric mean measured concentrations:

Details on test conditions:

Test Vessel Preparation
Ten replicate test vessels were prepared for the control and each test concentration. A volume of ca 50 mL of the appropriate test or control medium was added to each vessel.
One juvenile Daphnia magna, less than 24 hours old, was added to each test vessel, using a wide bore glass pipette to avoid damaging the animals during transfer.
The media were renewed daily. Two sets of test vessels were used during the test. On each renewal day, new solutions were prepared and dispensed into a second vessel. The adult Daphnia were transferred across into the new vessel. The vessels were rinsed between media renewals. A wide bore glass pipette was used for the daphnia transfer.

Appearance of Test Media
The appearance, colour and behaviour of the test substance in the test media were recorded at daily intervals.

Feeding
The Daphnia magna were fed daily with a suspension of Chlorella vulgaris (CCAP 211/12). The daily feeding rate of Chlorella vulgaris was between approximately 1.987 x 107 and 3.987 x 107 cells per vessel per day to provide each Daphnia magna with between approximately 0.1 and 0.2 mg carbon/day (cell concentration ranges were calculated using recent total organic carbon (TOC) analysis data for the Chlorella vulgaris culture (10 December 2020)).
The amount of algae added to each test vessel was based on the age of the Daphnia magna and on the amount of precipitated algae present on the base of the vessels or in suspension directly before feeding.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 5.81 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

no effect also found in adult survival, adult growth and time first brood

Duration:

21 d

Dose descriptor:

other: EC20

Effect conc.:

> 5.81 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

no effect also found in adult survival, adult growth and time first brood

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 5.81 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

no effect also found in adult survival, adult growth and time first brood

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

5.81 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

no effect also found in adult survival, adult growth and time first brood

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 5.81 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

no effect also found in adult survival, adult growth and time first brood

Range-Finding Test

Analysis of the test media samples was conducted at the start and end of the first renewal period and has been summarised in the table below.

Nominal Concentration (mg/L)		Measured Concentration (mg/L)		Nominal Percentage Recovery (%)	% Recovery from Initial Measured	Geometric Mean Measured Concentration (mg/L)^A
		Day 2	Day 5	Day 2	Day 5
Control		ND	ND	NC	ND	NC
0.01		<LOQ (0.00865)	ND	86.5	ND	0.00500
0.1		0.112	ND	112	ND	0.0167
1		1.11	<LOQ (0.003)	111	<LOQ (0.3)	0.149
10		11.7	5.84	117	49.8	8.28

ND. Not detected below lowest calibration standard (0.005 mg/L)

NC. Not calculated

<LOQ. Below limit of quantification (lowest fortification standard - 0.02 mg/L)

^A. Calculated using either half the LOQ or half the lowest calibration standard where no reliable value present

Red.Red and italics = measured concentrations being outside the nominal range (80 – 120%)

The new media chemistry results indicated that the test substance was dosed correctly, as with exception to the 0.01 mg/L measured concentration which was below the lowest fortification standard so was not considered to be completely reliable, the new samples were within 80 – 120% of the nominal concentrations (111 - 117 % of nominal target).

The old media chemistry results after the 3-day renewal period indicated that the test substance was not stable over the media renewal because all measured concentrations except the highest concentration were below the lowest fortification standard. The highest concentration was also 49.8% of the initial measured concentration.

The mean number of live juveniles produced per treatment and parentalDaphnia magnasurvival by Day 13 (termination) are presented in the table below:

Nominal Concentration (mg/L)	Geometric Mean Measured Concentration (mg/L)	Adult Mortality (%)	Average Number of Juveniles per surviving adult	Coefficient of Variation of replicates (%)
Control	ND	0	25^A	NC
0.01	0.00500	0	41	13.9
0.1	0.0167	0	40	7.0
1	0.149	0	45	20.4

ND. Not detected

NC. Not calculated

^A. Vessel 1 excluded because it was slow to produce juveniles (first brood Day 12) and therefore, was out of synchronisation with the other vessels

The results of the range-finding test indicated that there were no test substance effects observed during the test and therefore, indicated that the NOEC was 10 mg/L and that the EC₅₀would be >10 mg/L.

The results of the range finding test also indicated that the test substance was not stable over the 3 day period assessed and therefore, the definitive test was conducted with daily media renewals.

Definitive Test

Chemical Analysisof Sodium Benzoate in the Test Media

The results of the chemical analysis of test media samples during the definitive test are presented in Table 1 (Attachment 1)

The LOQ for the analytical method was 0.02 mg/L.

Analysis of the test media samples was conducted at the start and end of three renewal periods during the test. Samples were taken for analysis of fresh or new solutions on Days 0, 11 and 20 and for analysis of the corresponding old media solutions on Days 1, 12 and 21.

The new media samples at Day 0 were all within the nominal target range (88.8 – 110% of the nominal target concentration).

The new media samples at Day 11 were initially 135 – 159% of nominal target, which were high. The “B” and “C” samples were analysed for confirmation. As there had been a delay before the initial samples were analysed (5 days after collection), the “B” and “C” samples had been stored for 8 days before being analysed and were outside the established storage stability period (7 days). The results of the “B” and “C” samples were variable and gave no conclusive result confirmation. In general (except the 3.2 mg/L treatment “B” and “C” results), the mean of the two samples were within 30% of the initial results and therefore, were considered to confirm the results. There was no toxicity at any of the test concentrations, so the 10 mg/L treatment was considered to be the most critical test level. Although variable (measured “B” sample result = 17.6 mg/L; measured “C” sample results = 8.36 mg/L), the mean of the two repeat samples for the 10 mg/L treatment was 13.0 mg/L which is within 20% of the initial measured result (measured “A” sample results = 15.9 mg/L), so confirms the initial result. A geometric mean measured concentration was used for reporting purposes, so this high result was not considered to have an impact on the overall results because it would normalise out the data.

With exception to the 1 and 3.2 mg/L treatments (77.3 and 70.0% of nominal target, respectively), the new media samples at Day 20 were all within the 80 – 120% nominal target (81.4 – 94.4% of nominal target). The slightly lower 1 and 3.2 mg/L treatments were not considered to have an impact on the results, because they were only slightly outside the nominal range (within 20% of the 10 mg/L measured concentration indicating they were prepared correctly) and also the 10 mg/L treatment was considered to be the critical treatment level, as there were no test substance effects at any of the test treatments.

The old media analysis indicated that the test substance was not stable over the 24‑hour test media renewal period. The stability appeared to be concentration dependent (more unstable at lower test concentrations) and worsened as the test progressed suggesting it was affected by a higher biological load. The majority of the old media concentrations for the 0.1 – 3.2 mg/L treatments were below the limit of quantification by the end of the 24-hour renewal (except Day 1 for the 1 and 3.2 mg/L treatments which were 52.8 and 79.4% of nominal respectively). The 10 mg/L old media sample recoveries from nominal were 91.1% on Day 1; 19.0% on Day 11 and 17.2% on Day 21.

Geometric mean measured concentrations have been used for presenting the results as they provide worst-case scenario and are considered to better normalise the change in degradation pattern observed over the test. The geometric mean concentrations were calculated to be 0.0406, 0.0603, 0.195, 0.437 and 5.81 mg/L, respectively.

Water Quality and Environmental Conditions

The results of the water quality determinations are summarised in the table below. All water quality and environmental measurements were within protocol specifications.

Parameter	Criterion	Observed Values^A
pH	6 – 9 (maintained within ± 1.5)	7.51 – 8.22 (max variation = 0.71 units)
Temperature (°C) – Actual test vessels (measurements at the start and end of each renewal)	18 - 22 (controlled within 2)	18.7 – 20.3°C (max variation = 1.6 )
Temperature (°C) – Continuously monitored	18 - 22 (controlled within 2)	20.2 – 21.1°C (max variation = 0.9)
Dissolved Oxygen (mg O2/L)	>3	7.50 – 9.45 mg/L
Light intensity (lux)	1000 – 1500	1007 – 1221 Lux

^A. Range represents the observed values across all vessels.

Test Media Descriptions

Test media observations were conducted on the new and old media on a daily basis. The observations were as follows:

• New Media - Colourless solutions in the control and test treatments for the duration of the test.

• Old Media - Colourless solutions with algae present in the bottom of the vessel in the control and test treatments for the duration of the test.

Survival of Daphnia magna

Parental Daphnia magna survival observed during the 21-Day definitive test is summarised in the table below:

Nominal Concentration (mg/L)	Survival of ParentalDaphnia magnaat Day 21 (%)
Control	100
0.1	90
0.32	90
1	90
3.2	80
10	90

The definitive test indicated no significant toxic effect on survival at the 10 mg/L nominal treatment (5.81 mg/L geometric mean measured concentration).

Daphnia magna Juvenile Production

Juvenile production per surviving adult, per test vessel, over the duration of the definitive test is summarised in the following table:

Nominal Concentration (mg/L)	Mean Cumulative Live Juvenile Production at Day 21 per surviving AdultD. magna	Standard Deviation	Coefficient of variation (%)
Control	106	26.8	25.3
0.1	104	25.4	24.4
0.32	116	39.9	34.4
1	111	22.2	20.0
3.2	135	27.3	20.2
10	111	25.9	23.3

The mean cumulative live juvenile production per surviving adult for each treatment is presented graphically in Figure1 (Attachment 2).

Any replicate where juveniles were produced, but the adult did not survive to Day 21 (one replicate at the 0.1, 0.32, 1 and 10 mg/L treatments and two replicates at the 3.2 mg/L treatment) or were identified to be male (one replicate at the 0.32 and 3.2 mg/L treatments) were excluded from the calculations, as recommended by the OECD 211 guideline.

The majority of the viable control and test vessels produced their first brood between Day 8 and 10. There were three vessels which were slow to produce (vessel 5 (Control), 18 (0.1 mg/L), 21 (0.32 mg/L) and 57 (10 mg/L) had their first broods on Day 15, 13, 16 and 12 respectively) and two males observed during the test. As there was also a delayed control replicate, the delay to first brood was not considered to indicate a treatment effect. This was confirmed by statistical analysis, as when the data was assessed for time to first brood, no significant toxic effects were observed. There were a few dead juveniles on Day 18 in control vessels 7, 8, 9 and 10 (24 dead juveniles recorded across the four vessels) and 3.2 mg/L treatment vessel 48 (5 dead juveniles recorded). The dead juvenile numbers and male daphnia were excluded from the results, so this did not have any impact on the test acceptance numbers. The number of dead juveniles were very low and occurred mostly in the control so this was not considered to be test substance related. These have been reported inTable5and indicated no treatment related trend. No aborted eggs were observed in the control or treatment groups throughout the test. The definitive test indicated no significant toxic effect on reproduction at the 10 mg/L nominal treatment (5.81 mg/L geometric mean measured concentration).

The mean cumulative live juvenile production per surviving adult for each treatment is presented graphically in Figure 1( Attachments 2)

Any replicate where juveniles were produced, but the adult did not survive to Day 21 (one replicate at the 0.1, 0.32, 1 and 10 mg/L treatments and two replicates at the 3.2 mg/L treatment) or were identified to be male (one replicate at the 0.32 and 3.2 mg/L treatments) were excluded from the calculations, as recommended by the OECD 211 guideline.

The majority of the viable control and test vessels produced their first brood between Day 8 and 10. There were three vessels which were slow to produce (vessel 5 (Control), 18 (0.1 mg/L), 21 (0.32 mg/L) and 57 (10 mg/L) had their first broods on Day 15, 13, 16 and 12 respectively) and two males observed during the test. As there was also a delayed control replicate, the delay to first brood was not considered to indicate a treatment effect. This was confirmed by statistical analysis, as when the data was assessed for time to first brood, no significant toxic effects were observed.

There were a few dead juveniles on Day 18 in control vessels 7, 8, 9 and 10 (24 dead juveniles recorded across the four vessels) and 3.2 mg/L treatment vessel 48 (5 dead juveniles recorded). The dead juvenile numbers and male daphnia were excluded from the results, so this did not have any impact on the test acceptance numbers. The number of dead juveniles were very low and occurred mostly in the control so this was not considered to be test substance related. These have been reported in Table 5 (Attachment 3) and indicated no treatment related trend.

No aborted eggs were observed in the control or treatment groups throughout the test.

The definitive test indicated no significant toxic effect on reproduction at the 10 mg/L nominal treatment (5.81 mg/L geometric mean measured concentration).

Growth Measurements

The results of the individual carapace measurements for the surviving adults are summarised in the table below:

Nominal Concentration (mg/L)	Mean Length ofDaphnia magnaat Day 21 (mm)
Control	4.0
0.1	4.0
0.32	3.9
1	4.0
3.2	4.1
10	4.0

The definitive test indicated no significant toxic effect on growth at the 10 mg/L nominal treatment (5.81 mg/L geometric mean measured concentration).

Toxicity Values

The 21-day EC₁₀, EC₂₀, EC₅₀, NOEC and LOEC toxicity values in terms of adult survival, live juveniles produced per survivingparental Daphnia magna, adult growth and time to first brood are presented based on geometric mean measured concentrations in the table below:

Parameter		Toxicity Value based on Geometric Mean Measured Concentrations (mg/L)
		Adult Survival	Juvenile Production	Adult Growth*	Time to first brood
EC10		>5.81^A	>5.81^A	>5.81^A	>5.81^A
EC20		>5.81^A	>5.81^A	>5.81^A	>5.81^A
EC50		>5.81^A	>5.81^A	>5.81^A	>5.81^A
NOEC		5.81	5.81	5.81	5.81
LOEC		>5.81	>5.81	>5.81	>5.81

*Based on carapace length

^AThis was empirically derived as no effects were noted when statistically assessed for the NOEC and LOEC parameters

Validity Criteria

The validity criteria were satisfied as detailed below:

- Daphnia magna in the control treatment was 0% (≤20%) by the end of the test.

- Daphnia magna in the control (female) at the end of the test was 106 (>60).

- No ephippia were observed in the controls during the test.

It was therefore considered that the results of the reproduction test are valid.

Validity criteria fulfilled:

yes

Conclusions:

The 21-Day EC10, EC20 and EC50 values in terms of Daphnia magna reproduction, survival, growth and time to first brood were >5.81 mg/L based on based geometric mean measured concentrations. This was empirically derived as no effects were noted when statistically assessed for the NOEC and LOEC parameters.

Executive summary:

A 21-day Daphnia magna reproduction study was conducted to determine the effects of exposure to Sodium Benzoate on Daphnia magna reproduction, survival, growth and time to first brood. In comparison to the control group, no significant effects at any concentration were observed for Daphnia magna reproduction, survival, growth and time to first brood, therefore, the NOEC and LOEC were considered to be 5.81 and >5.81 mg/L, respectively, based geometric mean measured concentrations.

The 21-Day EC_10,EC₂₀andEC₅₀values in terms of Daphnia magna reproduction, survival, growth and time to first brood were >5.81 mg/L based on based geometric mean measured concentrations. This was empirically derived as no effects were noted when statistically assessed for the NOEC and LOEC parameters.

The validity criteria were satisfied, and therefore the results of the reproduction test are considered to be valid.

Description of key information

The 21-Day NOEC and EC₅₀values in terms of Daphnia magna reproduction, survival, growth and time to first brood were 5.81 and >5.81 mg/L, respectively based on based geometric mean measured concentrations.  The validity criteria were satisfied, and therefore the results of the reproduction test are considered to be valid.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

5.81 mg/L

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

> 5.81 mg/L

Additional information

A 21-day Daphnia magna reproduction study was conducted to determine the effects of exposure to Sodium Benzoate on Daphnia magnareproduction, survival, growth and time to first brood.

In comparison to the control group, no significant effects at any concentration were observed forDaphnia magnar eproduction, survival, growth and time to first brood, therefore, the NOEC and LOEC were considered to be 5.81 and >5.81 mg/L, respectively, based geometric mean measured concentrations.