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Real Science



 

Coral Toxicity Experiment Using Reef Safe™ Sunscreen

Mote Marine Laboratory

Tropical Research Lab, Summerland Key, Florida, USA

February 26, 2015

Introduction: Coral reefs have been under stress from multiple factors over the last 3 decades.  Efforts have been made to identify the threats and lower the impacts. One such area of concern that has been debated recently is over the effects of sunscreens or their compounds on the health of corals. Various tests have shown conflicting results. This test is to introduce formulations of one brand of sunscreen, Reef Safe™, (RS) with two Atlantic hard corals for potential toxicity of the product formulations, which include the active ingredient oxybenzone (OB).

PurposeThe purpose of this experiment is to test RS sunscreen in an acute toxicity 48 hour LC 50 test with two species of Atlantic Corals. The experiment will test RS sunscreen formulations both without OB, then with OB at 4%, with OB at 6%, and with OB at 6% but administered at 29.84 times the quantity expected environmental concentrations (EEC).  If the tests show no observable differences, then it will be continued for another 48 hours to provide a 96 hour LC 50 test. This will be continued for additional continuous, 96 hour test periods until the experiment was terminated.

Methods:   Two species of hard corals were chosen because of their presence in inshore and offshore habitats and forms.  The offshore, massive coral, Orbicella faveolata, (O. fav), (mountainous star coral), was chosen as a reef building coral.  The inshore, branching coral, Porites, (P. por), (finger coral), was chosen as a representative of shallow inshore coral. Coral were fragmented into: 100 O. fav and 100 P. por fragments and attached to ceramic bases for ease of handling. Each of them was distributed equally between 20 tanks with 5 of each species in a given 20 liter aquarium. The corals are healed over and acclimated for two weeks prior to the start of the experiment. All tanks were placed into our temperature controlled raceway and supplied filtered seawater at a temperature of 27 °C under natural light conditions.  

Materials and Methods:   Raceways were set up with a total of 20 5-gallon tanks each randomly labeled either A, B, C, D, or E for the five treatments. Each tank contained 5 individuals of each species, O. fav and P. por. All tanks shared the same seawater with flow rates of 150 ml/min and temperature of 27C to negate any variation that may affect coral growth. Before starting the experiment each fragment was visually inspected to assess for health or mortality, to be compared up to 20 days or the completion of the experiment.  Corals were visually assessed using a coral health established by the Florida Fish and Wildlife Conservation Commission (FWC).

Plates were set onto a template that marked where the sunscreen should be applied. Product was smeared onto 12” x 12” x 0.25” tempered glass plates within a horizontal section 5.5 – 7.5 inches from the bottom using latex finger cots (cut from laboratory gloves). 

There are five treatments: Control/no product (C),  Oxybenzone-free SPF 30 formula (B), SPF 50 containing 4.064% Oxybenzone formula (E), SPF 45 containing 6.00 % oxybenzone formula (D), and SPF 45 6.00% Oxybenzone at  5 times the dosage (A). 10 +/- 0.01 mg of SPF 30, 50, and 45 formulas were added to four plates each, and 50 mg, ( 29.84 x EEC) of SPF 45 was added to an additional four plates. Plates were allowed to dry for at least 24 hours.   Additional active ingredients and formulary components are 7.5 % Octinoxate, 5.0 % Octisalate, 10.0%  Homosalate, 10.0% Octocrylene, 3.0% Avobenzone, including a preservative system for all formulas comprised of 0.25% Methylparaben, 0.10%  Propylparaben, 0.30%  Diazolidinyl urea. 

Each day before the sunscreen plates were added, a condition survey was completed to note which corals were showing signs of stress. Corals were ranked on a scale of 1-6 for condition (tissue loss) and color (signs of bleaching) taken from Monitoring Data Collections guidelines by Florida Fish and Wildlife Conservation Commission (FWC) Health Monitoring Protocols. The corals were assessed using the following scoring conditions:

  • 1. No live tissue
  • 2. <25% live tissue
  • 3. 25-50% live tissue
  • 4. 50-75% live tissue
  • 5. 75-95% live tissue
  • 6. No apparent loss

Twice a day (10a and 2p) the dosed plates were set into the corresponding research tanks for 60 minutes, after which the plates were removed and water flow was immediately returned. During the 1 hour treatment all external water flow to all tanks was stopped, but continuing to allow the powerhead to circulate water past the glass plate and thru the tank and corals. After the 1 hour treatment period water flows were returned to normal allowing for a gradual delusion of the tank water with new water. The Glass plates were washed thoroughly using Reef Safe™ soap and nanopure water and set out to dry before new product was applied again. 

CORAL REEF SAFE SUNSCREEN PLATE GLASS CLEANING AND PLATING SEQUENCE

Treatments are as follows:

  1. SPF 45 high @ 50mg test formula id 50-0045-15 batch # 100
  2. Blank (SPF 30 oxybenzone free) @ 10mg test formula id 50-0300-13 batch # 102
  3. Control (glass slide only, no lotion)
  4. SPF 45 @ 10mg test formula id 50-0045-15 batch # 100
  5. SPF 50 @ 10mg test formula id 50-0500-15 batch # 101

3.0 Control glass plates

OB-3 concentrations in treatments:

  1. SPF 45 HIGH @ 50mg TEST FORMULA ID 50-0045-15 BATCH # 100
    • 97 µg/L
    • EEC 5.67 µg/L
    • 84x greater than average reef exposure
  2. BLANK (SPF 30 OXYBENZONE FREE) @ 10mg TEST FORMULA ID 50-0300-13 BATCH # 102
  3. CONTROL (GLASS SLIDE ONLY, NO LOTION)
  4. SPF 45 @ 10mg TEST FORMULA ID 50-0045-15 BATCH # 100
    • 80 µg/L
    • EEC 1.13 µg/L
    • 95x greater than average reef exposure
  5. SPF 50 @ 10mg TEST FORMULA ID 50-0500-15 BATCH # 101
    • 20 µg/L
    • EEC 0.76 µg/L
    • 00x greater than average reef exposure

OB-3 concentrations determined by dividing the dosage by volume in treatment tank (15.875L) and multiplying by the percentage of lotion represented by oxybenzone (6% in SPF 45 and 4% in SPF 50). EEC dosage determined from an independent study determining that approximately 3% of Reef Safe sunscreens enter the environment. Data from the Florida Keys suggests that Reef Safe’s™ EEC after a snorkel trip with 100 people in the water is 0.00314 mg/L of lotion, representing 0.19 µg/L of oxybenzone. Therefore, the treatments described above represent far greater chemical contributions to seawater than would be experienced on a given reef.

RESULTS:

Results for the first 48 hour test showed no differences between any level of sunscreen treatments and the control. The test was continued to perform a 96 hour test which also showed no differences. The experiment was extended for the duration of five 96 hour continuous treatments for a total duration of 20 days. No coral fragments of either species were lost during this experimental period. At the end of the 20 day experiment, there were no mortalities and no significant difference observed at any level of exposure of test fragments relative to controls.

In conclusion, the varying Reef Safe sunscreen formulations used in this test showed no mortalities or significant tissue loss on the corals tested over a period of 24hrs, 96hrs, and 20 days, using a 2x daily exposure testing protocol. This testing protocol was designed to simulate conditions found at coral reef sites impacted by large volumes of bathers and divers wearing EEC of Reef Safe ™ formulated sunscreen.

Chemistry Analysis

There was no significant difference in coral health for fragments grown in the different treatment conditions. Using an alpha of 0.05 for significance, a repeated measures ANOVA showed that there were no significant differences across time for the condition score of corals in each treatment condition (Table 1). The times used for this analysis were T= 0 hours, 24 hours, 120 hours, and 456 hours, which was the final day of observations. This data was run with condition score for each coral as the response, time as the x factor, and separated by treatment condition.

Figure 1: Sum of condition scores for corals in each treatment plotted across time. Linear slopes indicate that there was very little change in condition (tissue cover) for fragments growing in each of the five treatment conditions.

Table 1: Repeated measures ANOVA results for condition scores of each coral fragment in the five treatment conditions. There was no significant difference across time for corals in any treatment (α > 0.05).

Treatment

N

ChiSquare

Prob > ChiSquare

Control (C)

40

0

1.0000

Blank (B)

40

0.03

0.8696

SPF 45 50 mg (A)

40

0.08

0.7748

SPF 45 10 mg (D)

40

0.01

0.9433

SPF 50 10 mg (E)

40

0.13

0.7226

 

DISCUSSIONS: This experimental test of sunscreen treatments on coral health showed no mortality or any significant differences (.05) in condition relative to controls during 48 hour, 96 hour and 20 day duration LC50 tests. This experiment, unlike many others, used sunscreen concentrations representative of high usage diving, and bather events over coral reefs, similar to condition on heavily utilized coral reef dive sites. The test condition simulated normal sunscreen applications to a surface and did not use any solvents or dispersants to force the products into artificial solutions. The RS sunscreen formulation was also tested at concentrations 5 times the expected environmental concentration, exhibiting no visual impacts or mortality on the two representative species of live corals during tests of 48 hours, 96 hour or 20 days duration. This was also the case for comparison to the basic sun screen formulations without OB, indicating no mortality effects from any of the other ingredients in the RS formulations in the tested concentrations, which are 29.84 x EEC), as well as seawater control with no sunscreen. Chemical analysis of concentrations inclusive of OB in the water showed that that about 50% of the total OB in the 10mg of sunscreen was dispersed into the water (Table 2). The higher dose of 50mg sunscreen exhibited less % dispersion (32%), This experiment showed no mortality or any significant tissue or health change (.050) in visual condition using any of these formulas or concentrations on two species of Atlantic corals for both acute (48 hour and 96 hour LC50) or chronic 20 day duration.

Analysis of Oxybenzone, a component in Reef Safe™ sunscreen formulations, in Seawater from Coral Exposure Studies:

Analytical Methods:

Oxybenzone standard was purchased from Spectrum Chemical, dissolved in methanol and analyzed by high performance Liquid Chromatography, interfaced with tandem masspectrometric detection (HPLC-MS/MS) to develop the appropriate analytical method and establish the working calibration curve for the expected concentration range. Extraction efficiency was determined by adding a known amount of oxybenzone to seawater and extracting with dichloromethane (DCM). DCM was evaporated and replaced with simultaneous evaporation of methanol in preparation for LC-MS analysis.

Water samples were collected from coral exposure chambers on two occasions; 11/19/15 and 12/03/15. The first set collected 400ml into 500ml pre-cleaned glass jars with Teflon-lined caps.

50ml DCM was added to each jar, shaken vigorously to initiate oxybenzone extraction and inhibit degradation during transport. Samples were shipped overnight to the Mote Sarasota for further processing and HPLC-MS analysis. Samples collected on 12/02/15 were collected in 250ml glass bottles, 25 ml DCM added, shaken and shipped as above. Chain of custody was verified with custody sheets accompanying each set of samples.

Analysis of oxybenzone in seawater samples was performed using a Thermo Electron Corp. TSQ Quantum Access/ESI/Accela UHPLC for LC-MS/MS, interfaced with a Quantum Access triple quadrupole mass spectrometer. All analyses were conducted with an analytical column of hypersil gold C-18 (ThermoFischer), reverse phase, 5um particle size with dimensions of 100mm x 2.1mm and MeOH/H2O solvent gradient.

Results and Discussion:

Testing of the DCM extraction and HPLC-MS/MS analytical procedures exhibited a recovery mean ± standard deviation (n=3) of 87 ± 9 % for standard oxybenzone in seawater. These results provide verification of the methodology.

Sample designations:

Cont. = seawater control

Blank = reagent blank, Reef Safe™ sunscreen formulation with no oxybenzone

10mg 4% = 10mg of Reef Safe sunscreen formulation containing 4% oxybenzone

10mg 6% = 10mg of Reef Safe sunscreen formulation containing 6% oxybenzone

50mg 6% = 50mg of Reef Safe sunscreen formulation containing 6% oxybenzone

Results of the 11/19/15 exposure samples are given in Fig. 2, showing concentrations observed in each of two replicate samples from each exposure concentration. Concentrations of oxybenzone in the exposure chambers reflects the amount of oxybenzone applied to each glass plate, exhibiting 10 µg/L, 20 µg/L and 50 µg/L for the 10 mg 4%, 10 mg 6% and 50 mg 6% sunscreens, respectively.

Results of the 12/03/15 exposure samples are given in Fig. 3, showing concentrations observed in each of three replicate samples from each exposure concentration. Concentrations of oxybenzone in the exposure chambers reflects the amount of oxybenzone applied to each glass plate, exhibiting 11 µg/L, 20 µg/L and 65 µg/L for the 10 mg 4%, 10 mg 6% and 50 mg 6% sunscreens, respectively.

The mean and standard deviation of oxybenzone concentrations from all exposure chambers that were sampled is shown in Table 1. These data exhibit excellent precision among the 10mg replicate exposure chambers. The 50mg samples exhibited less, yet still acceptable, precision most likely due to the high amount of sunscreen collected at the surface of the tank. Table 1 also shows the calculated amount of oxybenzone dispersed into the water and the % of oxybenzone dispersed from the 10mg and 50mg samples of sunscreen spread on the glass plates.

Previous studies reported concentrations of oxybenzone to range from 75 to 1,400 µg/L in the US Virgin Islands, and from 0.8 to 19.2 µg/L in the Hawaiian Islands (Downs, et al. 2015). The aqueous concentrations from this test are well within the range observed for the Hawaiian Islands. The percentage of oxybenzone dispersed from the sunscreen spread on glass plates was calculated to be about 50% of the 10 mg samples (46% of 10-4% and 56% of 10-6%). The higher dose of 50 mg exhibited less % dispersion, most likely due to the limited solubility of the sunscreen and oxybenzone in water.

Table 2: Mean and standard deviation of oxybenzone (OB) µg/L comparing all samples in coral exposure chambers from both sampling dates. Including the calculated amount of OB in the 10 mg and 50 mg of sunscreen spread on the glass plates, the total amount of OB calculated to be in the 20 L exposure aquaria, and the calculated % of the OB that was dispersed from the 10 mg and 50 mg sunscreen samples.

 

µg/L OB in water

µg OB

µg OB

% OB

sample

mean

stdev

in sunscreen

in 15.9L aquaria

dispersed

Cont

0.22

0.06

NA

NA

NA

RB

0.09

0.13

NA

NA

NA

10-4%

11.71

2.02

400

186

46.6%

10-6%

21.30

2.60

600

339

56.5%

50-6%

60.67

13.46

3,000

965

32.2%